Analytical specificity and sensitivity of the novel dual‐target GeneProof Neisseria gonorrhoeae PCR kit for detection of N. gonorrhoeae

Detection of Neisseria gonorrhoeae relies increasingly on nucleic acid amplification tests (NAATs). The specificity of many gonococcal NAATs has been suboptimal and supplementary testing remains recommended in Europe and several additional countries. The novel dual‐target GeneProof Neisseria gonorrhoeae PCR kit, targeting porA pseudogene and 16S rRNA gene, showed a high specificity and sensitivity when isolates of non‐gonococcal Neisseria and related species (n = 144), and gonococci (n = 104) were tested. However, rare gonococcal porA mutants were only detected in the 16S rRNA gene target and two non‐gonococcal isolates showed a low‐level cross‐reactivity in the 16S rRNA gene target. The detection limit for both targets was 1.5 copies per reaction.     

Evaluation of a ten-minute chromogenic substrate test for identification of pathogenicNeisseria species andBranhamella catarrhalis

A ten-minute chromogenic substrate test was evaluated for its ability to rapidly identify pathogenicNeisseria spp. andBranhamella catarrhalis. Identifications obtained with this system were compared to those obtained using conventional procedures. The test correctly identified 98.9% of 90Neisseria gonorrhoeae, 98.3% of 60Neisseria meningitidis, 96.2% of 26Neisseria lactamica, and 100% of 36Branhamella catarrhalis strains. EightNeisseria subflava strains that grew on modified Thayer-Martin agar were prolyl aminopeptidase positive and were misidentified asNeisseria gonorrhoeae. Other strains of saprophyticNeisseria spp. also reacted with the chromogenic substrates. The system was accurate and reliable for identifying the commonly encountered pathogenic species. In light of recent reports describing new species and atypicalNeisseria strains, however, careful attention to the salient features of both common and atypical organisms is necessary for proper use of rapid enzymatic identification tests.     

Novel Genus-Specific PCR-Based Assays for Rapid Identification of Neisseria Species and Neisseria meningitidis

 This study presents the development ofpolymerase chain reaction (PCR)-based tests for the identification and detection of Neisseria species and Neisseria meningitidis. Currently, isolating and identifying these pathogens using conventional biochemical methods require 48–72 h. To improve speed and accuracy in diagnosing Neisseria infections, simple PCR-based tests that are specific for the genus Neisseria and the species Neisseria meningitidis have been developed. The genus-specific and species-specific DNA sequences were chosen by selecting and analyzing available database sequences. Neisseria-specific and Neisseria meningitidis-specific primer pairs were derived from the genes asd (coding for the aspartate β-semialdehyde dehydrogenase) and ctrA (coding for a conserved outer membrane protein), respectively. Both the Neisseria-specific and Neisseria meningitidis-specific PCR assays were specific (they amplified only DNA from the target genus or species, out of 84 bacterial species tested). In addition, the Neisseria-specific assay amplified DNA from 321 of 322 strains tested representing 13 species of Neisseria, while the Neisseria meningitidis-specific assay amplified DNA from all 256 strains tested representing nine serogroups of Neisseria meningitidis. These PCR assays, which can be combined in multiplex, have been adapted to ensure that they are simple and can be performed within approximately 90 min. The tests provide new diagnostic tools for identifying Neisseria infections.     

Multicenter evaluation of the new Vitek 2 Neisseria-Haemophilus identification card

The new Neisseria-Haemophilus identification (NH) card for Vitek 2 was compared with 16S rRNA gene sequencing (16S) as the reference method for accurate identification of Neisseria spp., Haemophilus spp., and other fastidious gram-negative bacteria. Testing was performed on the Vitek 2 XL system with modified software at three clinical trial laboratories. Reproducibility was determined with nine ATCC quality control strains tested 20 times over a minimum of 10 days at all three sites. A challenge set of 30 strains with known identifications and 371 recent fresh and frozen clinical isolates were also tested. Expected positive and negative biochemical reactions were also evaluated for substrate reproducibility. All microorganisms were tested on the NH card, and all clinical and stock isolates were saved for 16S testing. All reproducibility tests yielded expected results within a 95% confidence interval. For challenge microorganisms, there was 98% overall correct identification, including 8% low discrimination, 2% incorrect identification, and 0% unidentified. For clinical strains, there was 96.5% overall correct identification, including 10.2% low discrimination, 2.7% incorrect identification, and 0.8% unidentified. The 2.7% (10/371) of clinical isolates that gave an incorrect identification consisted of 7 isolates correct to genus and 3 strains incorrect to genus. There were an additional 27 strains (primarily Neisseria species) for which the 16S identification result was different from the NH card result. These were all unclaimed species by the system. The new NH card met all performance criteria within a 95% confidence interval compared to identification of clinical isolates by 16S.     

Sequence diversity of Neisseria meningitidis 16S rRNA genes and use of 16S rRNA gene sequencing as a molecular subtyping tool

We investigated the diversity of the primary sequences of 16S rRNA genes among Neisseria meningitidis strains (Men) and evaluated the use of this approach as a molecular subtyping tool. We aligned and compared a 1,417-bp fragment of the 16S rRNA gene from 264 Men strains of serogroups A, B, C, and Y (MenA, MenB, MenC, and MenY, respectively) isolated throughout the world over a 30-year period. Thirty-one positions of difference were found among 49 16S types: differences between types ranged from 1 to 14 positions (0.07 to 0.95%). 16S types and serogroups were highly associated; only 3 out 49 16S types were shared by two or more serogroups. We have identified 16S types that are exclusively associated with strains of certain hypervirulent clones: 16S type 5 with MenA subgroup III, 16S type 4 with the MenB electrophoretic type 5 (ET-5) complex, and 16S types 12 and 13 with MenC of the ET-37 complex. For MenC strains, 16S sequencing provided the highest sensitivity and specificity and the best overall association with the outbreak-related versus sporadic isolates when compared with pulsed-field gel electrophoresis, multilocus enzyme electrophoresis, and multilocus sequence typing. We demonstrated for the first time an unexpected diversity among 16S rRNA genes of Men strains, identified 16S types associated with well-defined hypervirulent clones, and showed the potential of this approach to rapidly identify virulent strains associated with outbreaks and/or an increased incidence of sporadic disease.     

Distribution of 16S rRNA methylases among different species of Gram-negative bacilli with high-level resistance to aminoglycosides

16S rRNA methylases confer high-level resistance to most aminoglycosides in Gram-negative bacteria. Seven 16S rRNA methylase genes, armA, rmtA, rmtB, rmtC, rmtD, rmtE and npmA, have been identified since 2003. We studied the distribution of methylase genes in more than 200 aminoglycoside-resistant Gram-negative clinical isolates collected in 2007 at our hospital in Shanghai, China. 16S rRNA methylase genes were amplified by polymerase chain reaction (PCR) among 217 consecutive clinical isolates of Gram-negative bacilli resistant to gentamicin and amikacin by a disk diffusion method. 16S rRNA methylase genes were present in 97.5% (193/198) of clinical isolates highly resistant to amikacin (≥512 μg/ml), with armA and rmtB detected in 67.2 and 30.3% of strains, respectively, while no 16S rRNA methylase genes were detected in 19 strains with amikacin minimum inhibitory concentration (MIC) ≤256 μg/ml. armA or rmtB genes were detected in 100% of 104 strains of Enterobacteriaceae, and these two genes were equally represented (49 vs. 55 strains). Genes for armA or rmtB were detected in 94.7% (89/94) of Acinetobacter baumannii and Pseudomonas aeruginosa strains, and armA was predominant (84 vs. 5 strains with rmtB). No rmtA, rmtC, rmtD or npmA genes were found. Enterobacterial repetitive intergenic consensus sequence (ERIC-PCR) indicated that armA and rmtB genes were spread by both horizontal transfer and clonal dissemination.     

Morphological and genetic diversity of symbiotic cyanobacteria from cycads

The morphological and genetic diversity of cyanobacteria associated with cycads was examined using PCR amplification techniques and 16S rRNA gene sequence analysis. Eighteen symbiotic cyanobacteria were isolated from different cycad species. One of the symbiotic isolates was a species of Calothrix, a genus not previously reported to form symbioses with Cycadaceae family, and the remainder were Nostoc spp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the repetitive sequences. Based on fingerprint patterns and phenograms, it was revealed that cyanobacterial symbionts exhibit important genetic diversity among host plants, both within and between cycad populations. A phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that most of the symbiotic cyanobacterial isolates fell into well‐separated clades. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Description of an Unusual Neisseria meningitidis Isolate Containing and Expressing Neisseria gonorrhoeae-Specific 16S rRNA Gene Sequences

An apparently rare Neisseria meningitidis isolate containing one copy of a Neisseria gonorrhoeae 16S rRNA gene is described herein. This isolate was identified as N. meningitidis by biochemical identification methods but generated a positive signal with Gen-Probe Aptima assays for the detection of Neisseria gonorrhoeae. Direct 16S rRNA gene sequencing of the purified isolate revealed mixed bases in signature regions that allow for discrimination between N. meningitidis and N. gonorrhoeae. The mixed bases were resolved by sequencing individually PCR-amplified single copies of the genomic 16S rRNA gene. A total of 121 discrete sequences were obtained; 92 (76%) were N. meningitidis sequences, and 29 (24%) were N. gonorrhoeae sequences. Based on the ratio of species-specific sequences, the N. meningitidis strain seems to have replaced one of its four intrinsic 16S rRNA genes with the gonococcal gene. Fluorescence in situ hybridization (FISH) probes specific for meningococcal and gonococcal rRNA were used to demonstrate the expression of the rRNA genes. Interestingly, the clinical isolate described here expresses both N. meningitidis and N. gonorrhoeae 16S rRNA genes, as shown by positive FISH signals with both probes. This explains why the probes for N. gonorrhoeae in the Gen-Probe Aptima assays cross-react with this N. meningitidis isolate. The N. meningitidis isolate described must have obtained N. gonorrhoeae-specific DNA through interspecies recombination.     

Superoxol and aminopeptidase tests for identification of pathogenicNeisseria species andMoraxella (Branhamella) catarrhalis

The superoxol test, and prolyl aminopeptidase and gammaglutamyl aminopeptidase tests were evaluated for the detection of pathogenicNeisseria spp. using 317 strains ofNeisseriaceae. The superoxol test was positive for all 116 gonococci and 62Moraxella (Branhamella) catarrhalis strains, but also for three strains ofNeisseria meningitidis, one strain ofNeisseria lactamica and eight saprophytic neisseriae. When using strains grown on Thayer-Martin medium, the positive and negative predictive values of the superoxol test for the identification ofNeisseria gonorrhoeae were 96.7 % and 100 % respectively. Meningococci were the only neisseriae growing on Thayer-Martin medium that showed gamma-glutamyl aminopeptidase activity. The prolyl aminopeptidase test showed low specificity.     

Comparison of Different DNA Fingerprinting Techniques for Molecular Typing of Bartonella henselae Isolates

Seventeen isolates of Bartonella henselae from the region of Freiburg, Germany, obtained from blood cultures of domestic cats, were examined for their genetic heterogeneity. On the basis of different DNA fingerprinting methods, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP) PCR, and arbitrarily primed (AP)-PCR, three different variants were identified among the isolates (variants I to III). Variant I included 6 strains, variant II included 10 strains, and variant III included only one strain. By all methods used, the isolates could be clearly distinguished from the type strain, Houston-1, which was designated variant IV. A previously published type-specific amplification of 16S rDNA differentiated two types of the B. henselae isolates (16S rRNA types 1 and 2). The majority of the isolates (16 of 17), including all variants I and II, were 16S rRNA type 2. Only one isolate (variant III) and the Houston-1 strain (variant IV) comprised the 16S rRNA type 1. Comparison of the 16S rDNA sequences from one representative strain from each of the three variants (I to III) confirmed the results obtained by 16S rRNA type-specific PCR. The sequences from variant I and variant II were identical, whereas the sequence of variant III differed in three positions. All methods applied in this study allowed subtyping of the isolates. PFGE and ERIC-PCR provided the highest discriminatory potential for subtyping B. henselae strains, whereas AP-PCR with the M13 primer showed a very clear differentiation between the four variants. Our results suggest that the genetic heterogeneity of B. henselae strains is high. The methods applied were found useful for typing B. henselae isolates, providing tools for epidemiological and clinical follow-up studies.     

Characterization of human clinical isolates of Dietzia species previously misidentified as Rhodococcus equi

In this study, 16 human clinical isolates of Dietzia species previously misidentified as Rhodococcus equi were evaluated using phenotypic methods, including traditional and commercial (API Coryne) biochemical tests, antimicrobial susceptibility testing, and 16S rRNA gene and gyrB gene sequencing. Positive results for both the hydrolysis of adenine and Christie–Atkins–Munch–Petersen (CAMP) reaction allowed for differentiation between the Dietzia isolates and the type strain of Rhodococcus equi; however, traditional and commercial phenotypic profiles could not be used to reliably identify Dietzia species. The analysis of 16S rRNA gene and gyrB gene sequences could discriminate all Dietzia strains from the type strain of R. equi. Most Dietzia species had distinct 16S rRNA gene and gyrB gene sequences; however, the 16S rRNA gene sequences of the type strains of D. schimae and D. cercidiphylli were identical to D. maris and D. natronolimnaea, respectively. Based on comparative sequence analysis, five clinical isolates clustered with D. maris/D. schimae and nine with D. natronolimnaea/D. cercidiphylli. The two remaining isolates were found to be most closely related to the D. cinnamea/D. papillomatosis clade. Even though molecular analyses were not sufficiently discriminative to accurately identify all Dietzia species, the method was able to reliably identify isolates that were previously misidentified by phenotypic methods to the genus level.     

Demonstration of the absence of intervening sequences within 23S rRNA genes from Campylobacter lari

Cloning, sequencing and characterization of nearly full‐length 23S rRNA genes in 12 urease‐positive thermophilic Campylobacter (UPTC) isolates were carried out using two novel PCR primer pairs. Nucleotide sequences of the 23S rRNA genes from the 12 isolates were first shown not to carry any intervening sequences (IVSs) in both the 25 and 45 helix regions. Then, two PCR primer sets were designed in silico for amplification of the helix 25 and 45 regions within 23S rRNA gene sequences from Campylobacter lari. No IVSs were identified within the 23S rRNA genes among a total of 53 isolates of C. lari, following PCR amplification, TA cloning and sequencing procedures. Intact 23S rRNA was identified in all 65 C. lari isolates, resulting in no production of the fragmented 23S rRNA. These data suggest that C. lari may not have any opportunity to interact with any other source of IVSs until now, or has been unable to integrate IVSs into their own genomes. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Detection of bacteraemia in patients with fever and neutropenia using 16S rRNA gene amplification by polymerase chain reaction

Episodes of fever and neutropenia are common complications of treatment for cancer. The use of prophylactic and early empirical antibiotics has reduced mortality but decreases the sensitivity of diagnostic tests based on culture. The aim of this study was to determine the potential of a broad diagnostic approach (eubacterial) based on 16S rRNA gene amplification and sequencing to augment cultural methods of diagnosis of bacteraemia in patients with fever and neutropenia in a regional paediatric oncology centre. One hundred eleven patient-episodes of fever and neutropenia were evaluated during the study period, 17 of which were associated with positive blood cultures, as follows:Staphylococcus epidermidis (n=6 episodes),Enterococcus faecium (n=2),Streptococcus sanguis (n=3),Streptococcus mitis (n=3),Staphylococcus aureus (n=1),Micrococcus spp. (n=1), andStenotrophomonas maltophilia (n=1). Eubacterial polymerase chain reaction (PCR) detected bacterial DNA in nine of 11 blood culture-positive episodes for which a sample was available for PCR; the species identified by sequence analysis were identical to those derived from the conventional identification of the cultured isolates. Bacterial DNA was detected in 20 episodes (21 bacterial sequences) associated with negative blood cultures, 18 of which occurred in patients who were receiving antibiotics at the time of sample collection. The species presumptively identified by partial 16S rRNA gene sequencing were as follows:Pseudomonas spp. (n=6 episodes),Acinetobacter spp. (n=5),Escherichia spp. (n=3);Moraxella spp. (n=3);Staphylococcus spp. (n=2);Neisseria spp. (n=1); andBacillus spp. (n=1). The results of this study suggest that molecular techniques can augment cultural methods in the diagnosis of bacteraemia in patients who have been treated with antibiotics.     

Evaluation of the BactiCard <Emphasis Type="Italic">Neisseria</Emphasis> for Identification of Pathogenic <Emphasis Type="Italic">Neisseria</Emphasis> Species and <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis>

The BactiCard Neisseria (Remel, USA) is a chromogenic enzyme substrate system for identifying Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, and Moraxella catarrhalis. The identification system consists of a card with four test circles impregnated with chromogenic substrates for indoxyl butyrate esterase (IB), prolyl aminopeptidase (PRO), γ-glutamyl aminopeptidase (GLUT), and ?-galactosidase (BGAL). These substrates permit the identification of Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica, respectively. After hydration of the circles with buffer, colonies from growth on selective media or a subculture are applied to the four circles. IB and BGAL reactions are read for a blue-green color after 2 and 15 min, respectively. PRO and GLUT reactions are read at 15 min for a red color after addition of a developer reagent. Identifications obtained with the BactiCard Neisseria were compared with those obtained using conventional procedures for 558 isolates in a blinded fashion. The BactiCard Neisseria identified 100% of 254 Neisseria gonorrhoeae, 100% of 125 Neisseria meningitidis, 53 (98.2%) of 54 Neisseria lactamica, and 123 (98.4%) of 125 Moraxella catarrhalis isolates. The BactiCard Neisseria is an accurate and rapid system for identification of these microorganisms in the clinical laboratory. Electronic Publication     

Human vaginal Lactobacillus rhamnosus harbor mutation in 23S rRNA associated with erythromycin resistance

Little is known about the diversity and distribution of resistance determinants in human commensal bacteria. The aim of this study was to determine the molecular mechanism responsible for high-level erythromycin resistance among five human vaginal Lactobacillus rhamnosus isolates. PCR screening for the presence of ermA, ermB and ermC methylase genes revealed no determinants responsible for detected erythromycin resistance. Therefore, sequences of 23S rRNA genes from L. rhamnosus strains were studied by PCR–RFLP analysis and sequencing of 23S rRNA genes. According to the results, in all erythromycin-resistant L. rhamnosus strains, the presence of a A → G transition mutation at position 2058 was discovered. Additionally, the isolates exhibited heterozygosity for the A2058/G2058 mutation among 23S rRNA gene copies. Presumably, the greatest number of mutated 23S rRNA operons was observed for the L. rhamnosus BGHV1’ strain that also had the highest MIC for erythromycin (MIC = 2048 μg mL⁻¹). This study reports the presence of transition mutations in the V region of 23S rRNA genes that most probably account for high-level erythromycin resistance observed for the first time in human vaginal lactobacilli.     

Endocarditis Due to Neisseria bacilliformis in a Patient with a Bicuspid Aortic Valve

We report a case of endocarditis due to the rod-shaped Neisseria species Neisseria bacilliformis. The phenotypic characterization of this recently characterized bacteria is difficult, and the identification requires the sequencing of the 16S rRNA gene. The resolution of the disease was complete after appropriate antibiotic therapy, and surgery was not required.     

A new confirmatory <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> real-time PCR assay targeting the porA pseudogene

The Roche Cobas Amplicor system is widely used for the detection of Neisseria gonorrhoeae but is known to cross react with some commensal Neisseria spp. Therefore, a confirmatory test is required. The most common target for confirmatory tests is the cppB gene of N. gonorrhoeae. However, the cppB gene is also present in other Neisseria spp. and is absent in some N. gonorrhoeae isolates. As a result, laboratories targeting this gene run the risk of obtaining both false-positive and false-negative results. In the study presented here, a newly developed N. gonorrhoeae LightCycler assay (NGpapLC) targeting the N. gonorrhoeae porA pseudogene was tested. The NGpapLC assay was used to test 282 clinical samples, and the results were compared to those obtained using a testing algorithm combining the Cobas Amplicor System (Roche Diagnostics, Sydney, Australia) and an in-house LightCycler assay targeting the cppB gene (cppB-LC). In addition, the specificity of the NGpapLC assay was investigated by testing a broad panel of bacteria including isolates of several Neisseria spp. The NGpapLC assay proved to have comparable clinical sensitivity to the cppB-LC assay. In addition, testing of the bacterial panel showed the NGpapLC assay to be highly specific for N. gonorrhoeae DNA. The results of this study show the NGpapLC assay is a suitable alternative to the cppB-LC assay for confirmation of N. gonorrhoeae-positive results obtained with Cobas Amplicor.     

Identifying Neisseria Species by Use of the 50S Ribosomal Protein L6 (rplF) Gene

The comparison of 16S rRNA gene sequences is widely used to differentiate bacteria; however, this gene can lack resolution among closely related but distinct members of the same genus. This is a problem in clinical situations in those genera, such as Neisseria, where some species are associated with disease while others are not. Here, we identified and validated an alternative genetic target common to all Neisseria species which can be readily sequenced to provide an assay that rapidly and accurately discriminates among members of the genus. Ribosomal multilocus sequence typing (rMLST) using ribosomal protein genes has been shown to unambiguously identify these bacteria. The PubMLST Neisseria database (http://pubmlst.org/neisseria/) was queried to extract the 53 ribosomal protein gene sequences from 44 genomes from diverse species. Phylogenies reconstructed from these genes were examined, and a single 413-bp fragment of the 50S ribosomal protein L6 (rplF) gene was identified which produced a phylogeny that was congruent with the phylogeny reconstructed from concatenated ribosomal protein genes. Primers that enabled the amplification and direct sequencing of the rplF gene fragment were designed to validate the assay in vitro and in silico. Allele sequences were defined for the gene fragment, associated with particular species names, and stored on the PubMLST Neisseria database, providing a curated electronic resource. This approach provides an alternative to 16S rRNA gene sequencing, which can be readily replicated for other organisms for which more resolution is required, and it has potential applications in high-resolution metagenomic studies.     

Use of 16S rRNA gene sequencing for rapid confirmatory identification of Brucella isolates

Members of the genus Brucella are categorized as biothreat agents and pose a hazard for both humans and animals. Current identification methods rely on biochemical tests that may require up to 7 days for results. We sequenced the 16S rRNA genes of 65 Brucella strains along with 17 related strains likely to present a differential diagnostic challenge. All Brucella 16S rRNA gene sequences were determined to be identical and were clearly different from the 17 related strains, suggesting that 16S rRNA gene sequencing is a reliable tool for rapid genus-level identification of Brucella spp. and their differentiation from closely related organisms.     

Species-Level Identification of Actinomyces Isolates Causing Invasive Infections: Multiyear Comparison of Vitek MS (Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry) to Partial Sequencing of the 16S rRNA Gene

Actinomyces species are uncommon but important causes of invasive infections. The ability of our regional clinical microbiology laboratory to report species-level identification of Actinomyces relied on molecular identification by partial sequencing of the 16S ribosomal gene prior to the implementation of the Vitek MS (matrix-assisted laser desorption ionization–time of flight mass spectrometry [MALDI-TOF MS]) system. We compared the use of the Vitek MS to that of 16S rRNA gene sequencing for reliable species-level identification of invasive infections caused by Actinomyces spp. because limited data had been published for this important genera. A total of 115 cases of Actinomyces spp., either alone or as part of a polymicrobial infection, were diagnosed between 2011 and 2014. Actinomyces spp. were considered the principal pathogen in bloodstream infections (n = 17, 15%), in skin and soft tissue abscesses (n = 25, 22%), and in pulmonary (n = 26, 23%), bone (n = 27, 23%), intraabdominal (n = 16, 14%), and central nervous system (n = 4, 3%) infections. Compared to sequencing and identification from the SmartGene Integrated Database Network System (IDNS), Vitek MS identified 47/115 (41%) isolates to the correct species and 10 (9%) isolates to the correct genus. However, the Vitek MS was unable to provide identification for 43 (37%) isolates while 15 (13%) had discordant results. Phylogenetic analyses of the 16S rRNA sequences demonstrate high diversity in recovered Actinomyces spp. and provide additional information to compare/confirm discordant identifications between MALDI-TOF and 16S rRNA gene sequences. This study highlights the diversity of clinically relevant Actinomyces spp. and provides an important typing comparison. Based on our analysis, 16S rRNA gene sequencing should be used to rapidly identify Actinomyces spp. until MALDI-TOF databases are optimized.