Identification of HACEK clinical isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid and accurate tool for the identification of many microorganisms. We assessed this technology for the identification of 103 Haemophilus parainfluenzae, Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae (HACEK) clinical isolates and 20 Haemophilus influenzae clinical isolates. Ninety-three percent of HACEK organisms were identified correctly to the genus level using the Bruker database, and 100% were identified to the genus level using a custom database that included clinical isolates.

Comparisons of subgingival microbial profiles of refractory periodontitis, severe periodontitis, and periodontal health using the human oral microbe identification microarray.

BACKGROUND: This study compared the subgingival microbiota of subjects with refractory periodontitis (RP) to those in subjects with treatable periodontitis (GRs = good responders) or periodontal health (PH) using the Human Oral Microbe Identification Microarray (HOMIM). METHODS: At baseline, subgingival plaque samples were taken from 47 subjects with periodontitis and 20 individuals with PH and analyzed for the presence of 300 species by HOMIM. The subjects with periodontitis were classified as having RP (n = 17) based on mean attachment loss (AL) and/or more than three sites with AL >or=2.5 mm after scaling and root planing, surgery, and systemically administered amoxicillin and metronidazole or as GRs (n = 30) based on mean attachment gain and no sites with AL >or=2.5 mm after treatment. Significant differences in taxa among the groups were sought using the Kruskal-Wallis and chi(2) tests. RESULTS: More species were detected in patients with disease (GR or RP) than in those without disease (PH). Subjects with RP were distinguished from GRs or those with PH by a significantly higher frequency of putative periodontal pathogens, such as Parvimonas micra (previously Peptostreptococcus micros or Micromonas micros), Campylobacter gracilis, Eubacterium nodatum, Selenomonas noxia, Tannerella forsythia (previously T. forsythensis), Porphyromonas gingivalis, Prevotella spp., Treponema spp., and Eikenella corrodens, as well as unusual species (Pseudoramibacter alactolyticus, TM7 spp. oral taxon [OT] 346/356, Bacteroidetes sp. OT 272/274, Solobacterium moorei, Desulfobulbus sp. OT 041, Brevundimonas diminuta, Sphaerocytophaga sp. OT 337, Shuttleworthia satelles, Filifactor alocis, Dialister invisus/pneumosintes, Granulicatella adiacens, Mogibacterium timidum, Veillonella atypica, Mycoplasma salivarium, Synergistes sp. cluster II, and Acidaminococcaceae [G-1] sp. OT 132/150/155/148/135) (P <0.05). Species that were more prevalent in subjects with PH than in patients with periodontitis included Actinomyces sp. OT 170, Actinomyces spp. cluster I, Capnocytophaga sputigena, Cardiobacterium hominis, Haemophilus parainfluenzae, Lautropia mirabilis, Propionibacterium propionicum, Rothia dentocariosa/mucilaginosa, and Streptococcus sanguinis (P <0.05). CONCLUSION: As determined by HOMIM, patients with RP presented a distinct microbial profile compared to patients in the GR and PH groups.

Frequency of putative periodontal pathogens among type 1 diabetes mellitus: a case-control study.

OBJECTIVE: The aim of the present study is to compare and assess the risk of periodontitis due to the presence of four putative periodontopathic bacteria viz., Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens. To fulfil the above objective, polymerase Chain reaction using the primers targeting 16S rRNA gene of the bacterial species was performed with the subgingival plaque collected from the permanent first molars of type 1 diabetic children and age matched healthy children. RESULTS: The prevalence of periodontal pathogens in diabetic and healthy children was 6% and 16% for E. corrodens, 18% and 36% for C. rectus, 2% and 2% for P. intermedia, 4% and 0%, for P. nigrescens respectively. Statistically, significant difference was not observed for the prevalence of all the four periodontal pathogens between type 1 diabetic and healthy children (P = 1.00). The results of the present study thus reveal a negative correlation of type I diabetes to periodontitis in association to Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens.

DNA-DNA hybridization demonstrates multiple bacteria in osteoradionecrosis.

Bone necrosis secondary to radiation was previously attributed to trauma of devitalized bone and microbiological sepsis. However, conventional microbiological technique has failed to demonstrate microorganisms throughout osteoradionecrotic bone, claimed to be hypoxic, hypovascular and hypocellular. The aim of the present study was to examine such bone for bacteria using DNA-DNA hybridization. Compared to standard culture methods this technique enables the investigation of a vast number of bacteria in a fairly short time. Twelve deep medullary specimens from resected radionecrotic mandibles were studied. A multitude of bacterial species were detected, most of them anaerobic. Porphyromonas gingivalis was the most predominant organism, followed by Fusobacterium nucleatum subspecies polymorphum. All samples contained Actinomyces, Prevotella and F. nucleatum. The results of this study indicate that bacteria, particularly anaerobes, may play a more fundamental role in the pathophysiology of osteoradionecrosis than being merely surface contaminants.

Current taxonomy and identification of nonfermentative gram negative bacilli.

In recent years significant advances have been made in the characterization and taxonomy of nonfermentative bacilli as well as in the recognition of their pathogenic potential for man. In addition, certain other infrequently isolated bacteria that may be fermentative have been recognized, and improved methods for their identification have been developed. This discussion attempts to present a practical approach to the identification of these micro-organisms in the clinical microbiology laboratory.

Haemophilus paraphrophilus infection: a pitfall in laboratory diagnosis.

Many clinical laboratories have difficulty in identifying a group of organisms which are catalase negative, oxidase positive, Gram negative rods. We describe a case of purulent sacroiliitis due to Haemophilus paraphrophilus where the organism was initially misidentified as Eikenella corrodens leading to inappropriate antimicrobial chemotherapy. We review the strains of H. paraphrophilus and E. corrodens that were identified by the National Collection of Type Cultures over the last ten years. Only 21 of 100 strains identified as E. corrodens were submitted as E. corrodens. Seven strains submitted as possible E. corrodens were identified as H. paraphrophilus. Several different species of Gram negative rods may produce pitting on agar and this seems to be poorly recognised. However, further tests are available to facilitate correct identification of these strains.

Eikenella corrodens in human oral and non-oral infections: a review.

There is substantial evidence in support of the existence of distinct clinical forms of human periodontal disease. Moreover, these different forms of periodontal disease may be associated with relatively distinct subgingival microflora, often involving microaerophilic or anaerobic Gram-negative bacterial species. Eikenella corrodens is a facultative Gram-negative bacillus which is a common inhabitant of the oral cavity and the intestinal and genital tracts. Its primary ecologic niche within the oral cavity appears to be dental plaque, both in periodontally healthy individuals and in periodontitis patients. However, E. corrodens is recognized as an infrequent human pathogen capable of causing extraoral infections, either as the sole infectious agent or as part of a mixed infection, its potential role in the etiology of periodontal disease is not well understood. E. corrodens is often present in the supra- and subgingival plaque of periodontally healthy subjects. On the basis of cross-sectional and longitudinal studies, E. corrodens appears to be somewhat more prevalent in subgingival plaque samples of periodontitis subjects than periodontally healthy individuals. However, the percentage of E. corrodens in the total cultivable microflora did not vary between the two groups. Microbiologic studies attempting to define the relationship between E. corrodens and periodontal disease assume that this species is essentially homogeneous and that all strains exhibit comparable pathogenic potential. However, E. corrodens exhibits 1) variable colony morphology, biochemical and serologic reactivity; 2) marked phenotypic diversity with respect to outer membrane protein and lipopolysaccharide structure; and 3) marked diversity in the restriction patterns of total genomic DNA. Thus, it is possible that a limited number of clones of E. corrodens may be associated with periodontal disease and/or extraoral infection, while other strains are relatively harmless commensals. Additional studies, possibly employing strain-specific nucleic acid probes, may be required to define the role of E. corrodens as a human periodontal pathogen.

Periodontopathic bacterial infection in childhood.

BACKGROUND: Little information is available on periodontopathic bacterial infection in childhood. We assessed the prevalence by age of 10 putative periodontopathic microorganisms in periodontally healthy children using a polymerase chain reaction (PCR) assay. METHODS: Plaque samples were collected from the buccal-mesial sulcus of the first molar or second primary molar in the right upper quadrant of 144 children (2 to 13 years old, 12 subjects from each year of age) who showed negligible periodontal inflammation. Using species-specific primers of Porphyromonas gingivalis, Bacteroides forsythus, Prevotella intermedia, Prevotella nigrescens, Campylobacter rectus, Eikenella corrodens, Actinobacillus actinomycetemcomitans, Capnocytophaga ochracea, Capnocytophaga sputigena, and Treponema denticola, PCR amplification was performed with bacterial genomic DNA from plaque samples. RESULTS: The results indicated that C. rectus, E. corrodens, A. actinomycetemcomitans, C. ochracea, and C. sputigena were found in about 50% of the plaque samples from all age groups, while B. forsythus and P. intermedia were detected less frequently, and P. gingivalis and T. denticola were not found. In contrast, the percentage of P. nigrescens-positive subjects increased with age in primary dentition, and reached about 50% at 7 years of age and older. Subject-based analyses suggested that the number of bacterial species in the plaque samples increased gradually with age until 5 years old, and then reached a plateau after the mixed dentition period. CONCLUSIONS: The colonization of many putative periodontopathic microorganisms can occur quite early in childhood without clinical signs of periodontal disease. However, colonization by P. gingivalis and T. denticola was not detected in periodontally healthy children.

DNA fingerprinting of Eikenella corrodens by pulsed-field gel electrophoresis.

In order to conduct molecular typing of Eikenella corrodens strains by macrorestriction fingerprinting, we evaluated different restriction enzymes for digestion of genomic DNA and determined the optimal parameters for separating E. corrodens DNA by pulsed-field gel electrophoresis. Ten E. corrodens strains isolated from oral and extraoral infection sites in different individuals were analyzed. The rare-cutting restriction endonucleases DraI, SmaI and XbaI usually used for pulsed-field gel electrophoresis analyses were not suitable for digestion of E. corrodens genomic DNA because they either did not digest the DNA or produced bands of similar molecular weights that could not be separated. Accordingly, among additional enzymes including BamHI, BglII, EcoRI and Hind III, we found BamHI and BglII to be the most suitable rare-cutting enzymes for pulsed-field gel electrophoresis analysis. They cleaved the genomes of all the above strains into 15-20 fragment bands that were clearly separated by the following pulsed-field gel electrophoresis conditions: 140 V with a running time of 40 h, pulse times of 5 to 50 s with linear ramping and an electrical field angle of 120 degrees. These conditions enabled us to distinguish 8 individual pulsed-field gel electrophoresis patterns from the 10 strains analyzed. However, only 4 identical outer membrane protein profiles were differentiated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data obtained in this analysis showed clonal divergence among members of the E. corrodens species, at the same time revealing this pulsed-field gel electrophoresis as being a highly attractive procedure for epidemiological investigation of this organism, including its acquisition and transmission.

Deoxyribonucleic acid relatedness and phenotypic variation among human isolates of Eikenella corrodens.

The type strain of Eikenella corrodens (Eiken 1958) Jackson and Goodman 1972 and eleven epidemiologically independent clinical isolates recovered from periodontal locations, putrid wounds, abscesses, and bacteraemias were investigated for their genomic relationships by DNA-DNA hybridization with the renaturation method, genome molecular complexity, DNA base composition and some phenotypic features. The bacterial strains studied were interrelated at or above the 80% DNA binding level, their chromosomal DNAs exhibiting a mean molecular mass of 1.7 x 10(9) daltons and a mean guanine plus cytosine content of 55.1 mol%. Variations in colonial morphology, hemolytic activity on sheep blood agar, reduction of nitrates, oxidation of carbohydrates, lipase, leucine, valine, and cystine aminopeptidase and acid phosphatase activities occurred among closely interrelated strains. The definition of the species and current identification keys must be emended accordingly.

Outer membrane protein and lipopolysaccharide heterogeneity among Eikenella corrodens isolates.

Outer membrane protein (OMP) and lipopolysaccharide (LPS) phenotypic diversity among 27 oral and extraoral strains of Eikenella corrodens was assessed by SDS-PAGE. Each strain exhibited one to three major protein bands in the 35- to 41.5-kDa range and one or two protein bands of lesser density in the 24.5- to 28-kDa range. Eleven OMP patterns were distinguished among the strains. While oral strains obtained from periodontally healthy and diseased subjects exhibited diverse OMP patterns, five of six strains from extraoral sites of infection expressed an identical OMP pattern. Comparison of the electrophoretic mobilities of LPS from these same strains revealed that E. corrodens LPS consists primarily of low apparent molecular mass forms. Sixteen different LPS phenotypes were differentiated among the strains, with no apparent correlation between LPS phenotype and clinical setting. Strains expressing the same OMP pattern frequently expressed variable LPS phenotypes and vice versa. Analysis of OMP or LPS pattern by SDS-PAGE may be useful in taxonomic and epidemiologic studies of E. corrodens. Additional studies assessing the potential influence of OMP composition on invasiveness of this organism appear warranted.

Characterization of Wolinella spp., Campylobacter concisus, Bacteroides gracilis, and Eikenella corrodens by polyacrylamide gel electrophoresis.

The small asaccharolytic, nonpigmenting gram-negative rods of the human oral cavity are difficult to differentiate from each other. Protein profiles of sonicated cells of Wolinella species, Campylobacter concisus, Bacteroides gracilis, and Eikenella corrodens were obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized with a silver stain. The gels were scanned with a laser densitometer, and the similarity of strains was computed by determining correlation coefficients of normalized densities along the gels. The strains were grouped by cluster analysis of the correlation coefficients. All species were distinct from each other. Several groups were found within E. corrodens. A colored silver stain was found to highlight species differences and appears to be useful in the rapid identification of fresh isolates.

A type-specific antigen of Eikenella corrodens is the major outer membrane protein.

Eikenella corrodens released antigenic material in the form of outer membrane fragments during growth. A culture filtrate antigen (AgA) from three strains reacted with antisera to whole bacteria with serotype specificity. A monoclonal antibody to the AgA of strain 1073 reacted with only 2 of 12 strains tested by enzyme-linked immunosorbent assay, immunofluorescence assay, and immunoelectrophoresis assay. The antigenic reactivity was resistant to boiling and to pronase and trypsin treatments. AgA was isolated from an outer membrane preparation after boiling in sodium dodecyl sulfate, radioiodinating the proteins, and precipitating AgA specifically with monoclonal antibody. A single protein band corresponding to the major outer membrane protein of E. corrodens was observed by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate. AgA had a molecular weight of 42,000, and its amino acid content resembled those reported for major outer membrane proteins with similar molecular weights from two other gram-negative organisms.

Catalase-positive Eikenella corrodens and Eikenella-like isolates of human and canine origin.

Ten catalase-positive isolates and one catalase-negative isolate that had been assigned to Eikenella corrodens were compared to the nomenclatural type strain regarding selected phenotypic and molecular features and chromosomal deoxyribonucleic acid (DNA) relatedness using the spectrophotometric method. Five catalase-positive human isolates were assigned to the genomic species Eikenella corrodens on the basis of high DNA relatedness levels. Three others, among them strain Chen UB 204, exhibited only moderate degrees of DNA relatedness to the type strain and with each other. Two catalase-positive isolates from dogs were closely interrelated, but yielded only low degrees of DNA binding with Eikenella corrodens and the Eikenella-like human isolates. These findings confirm that the human eikenellas comprise more than one genomic species and that the canine strains represent a distinct taxonomic entity. The differentiation of the strains investigated by conventional phenotypic features, hydrolytic enzyme reactions, and cellular carbohydrate patterns was considered.

Identification of Eikenella corrodens and Cardiobacterium hominis by genetic transformation.

Genetic transformation was employed to attain exact identification of Eikenella corrodens and Cardiobacterium hominis. The two species appeared both homogeneous and without genetic affinity to each other or to species of Kingella, Neisseria, and Moraxella, as tested for by rather sensitive procedures with streptomycin- and spectinomycin-resistance markers.

Variable serum immunoglobulin G immune response to genetically distinct Eikenella corrodens strains coexisting in the human oral cavity.

This study examined the variable serum immunoglobulin G (IgG) levels to genetically distinct autologous Eikenella corrodens strains by enzyme-linked immunosorbent assay (ELISA). Twenty subjects, including 10 adult periodontitis patients, 5 juvenile periodontitis patients and 5 periodontally healthy subjects were examined. Each subject was colonized by 2-8 genetically distinct E. corrodens strains. The serum IgG levels to autologous E. corrodens within individuals were significantly different in 7 adult periodontitis patients, 4 juvenile periodontitis patients and a periodontally healthy subject. Poor correlation was found in diseased subjects between serum IgG levels to autologous strains and to reference strains ATCC 23834 or FDC 373. Four adult periodontitis patients and two juvenile periodontitis patients exhibited significant serum IgG levels to autologous E. corrodens strains (two standard deviations above the mean for periodontally healthy subjects); two of these six diseased subjects exhibited low serum IgG levels to reference strains and would have been classified as low immune responders if only reference strains had been used in ELISA. This study showed the importance of using autologous E. corrodens strains in the assessment of serum IgG immune responses to this organism.

Clinical isolates of anaerobic gram-negative rods with a formate-fumarate energy metabolism: Bacteroides corrodens, Vibrio succionogenes, and unidentified strains.

Strains of anaerobic, gram-negative bacteria, isolated from human clinical specimens and from studies of human normal flora, that have energy metabolism similar to Vibrio succinogenes are described. Included are four human isolates of V. succinogenes, five similar strains of motile straight rods, three strains of Bacteroides corrodens, and two unidentified strains. All strains studied grew poorly in usual anaerobic broth media but produced good turbidity in overnight broth cultures in media containing fromate and fumarate, indicating that all have an energy metabolism similar to V. succinogenes: they gain energy by the transfer of electrons from formate or hydrogen to fumarate.

Clinical features of patients with invasive Eikenella corrodens infections and microbiological characteristics of the causative isolates.

Clinical features of 43 cases of invasive Eikenella corrodens infections diagnosed at National Taiwan University Hospital during a 6-year period (1993-1998) were analyzed. The clinical syndromes included head and neck infection (56%), pulmonary infection (23%), intra-abdominal infection (14%), cutaneous infection (5%), skeletal infection (2%), endocarditis (2%), and pelvic abscess (2%). Nearly two-thirds of the patients (63%) had pre-existing diseases. Malignancy (35%), especially of the head and neck, was the most common underlying illness. More than half of the patients (56%) had associated factors predisposing to invasive Eikenella corrodens infection. Polymicrobial infections occurred in 28 (65%) patients, with two-thirds of the concurrent isolates being streptococci (66%). Five cases were fatal, with four deaths directly attributable to invasive Eikenella corrodens infection. Antimicrobial susceptibility testing and molecular typing were performed on 23 preserved Eikenella corrodens isolates. Antimicrobial susceptibility testing showed that Eikenella corrodens isolates were susceptible to penicillin, amoxicillin, cefoxitin, cefotaxime, cefepime, ciprofloxacin, and imipenem. The isolates were resistant to clindamycin, metronidazole, cephalothin, and cefuroxime. None of the 23 isolates produced beta-lactamase. Random amplified polymorphic DNA patterns of the 23 isolates were different, suggesting that different clones of Eikenella corrodens caused these infections.