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.

Sixty-three clinical isolates of charcoal-black-pigmented, gram-positive coryneform rods were received for identification by the Centers for Disease Control and Prevention (CDC) and were provisionally designated CDC fermentative coryneform group 4 (FCG4). Forty-five of these were characterized by morphological, physiologic, antimicrobial susceptibility, cellular fatty acids, 16S rRNA gene sequencing, and DNA-DNA hybridization analyses. Nitrate reduction, cellular fatty acid analysis, 16S rRNA gene sequencing, and DNA-DNA hybridization studies segregated these strains into two groups: FCG4a (8 strains) and FCG4b (37 strains). The FCG4a strains, only one of which was from a female genitourinary source, produced cellular fatty acid and biochemical profiles similar to those observed with reference strains of Rothia dentocariosa and Rothia mucilaginosa, while the FCG4b strains were similar to Corynebacterium species. DNA-DNA hybridization analysis demonstrated species-level relatedness among six FCG4a tested strains and showed that they were a charcoal-black-pigmented variant of R. dentocariosa. Sixteen isolates of the FCG4b group, mainly from female genitourinary tract specimens, as well as the type strains of two recently named species, Corynebacterium aurimucosum and Corynebacterium nigricans, were shown by DNA-DNA hybridization analysis and the sequencing of the 16S rRNA gene to be related at the species level and unrelated to the type strain of R. dentocariosa; therefore, the Corynebacterium-like strains were classified as a charcoal-black-pigmented variant of C. aurimucosum, because this name has nomenclatural priority over C. nigricans. These findings indicate that FCG4 represents a heterogeneous group that contains pigmented variants of both R. dentocariosa and C. aurimucosum; hence, the descriptions of both R. dentocariosa and C. aurimucosum have been amended to include charcoal-black-pigmented variants, and C. nigricans is a pro synonym of C. aurimucosum.

A Pasteurella-like bacterium associated with pneumonia in captive megachiropterans.

A novel Pasteurella-like organism was recovered postmortem from lung tissue of two captive Wahlberg's epauleted fruit bats (Epomophorus wahlbergi), with severe, unilateral pneumonia. The bats had been recently shipped and died shortly after release from a 30-day quarantine. One presented with clinical signs of anorexia and lethargy before death; the other died without prior clinical symptoms. The same Pasteurella-like organism was recovered antemortem from subcutaneous abscesses in two captive little golden mantled flying foxes (Pteropus pumilus) housed with additional E. wahlbergi. The organism was also cultured on tracheal wash from one Malaysian flying fox (Pteropus vampyrus) and another E. wahlbergi, both demonstrating clinical signs of pneumonia. All recovered isolates appeared morphologically and biochemically similar to the initial isolates and were further characterized as either a Pasteurella or Actinobacillus organism on the basis of biochemical and cellular fatty acid profiles. Screening of the current collection using pharyngeal swabs isolated this organism from 12 of 15 E. wahlbergi, two of three P. vampyrus, one of 26 island flying foxes (Pteropus hypomelanus), and one of nine Rodrigues fruit bats (Pteropus rodricensis). The organism was not identified in pharyngeal culture from eight Indian flying foxes (Pteropus giganteus), nine Egyptian fruit bats (Rousettus aegypticus), or an additional 16 P. pumilus.

Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax.

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

Bordetella holmesii bacteremia: a newly recognized clinical entity among asplenic patients.

Bordetella holmesii is a recently identified gram-negative bacterial species associated with bacteremia, endocarditis, and respiratory illness, mainly in immunocompromised patients. From isolates submitted to the Centers for Disease Control and Prevention from 1983 through 2000 for further identification, we identified 30 patients with B. holmesii bacteremia. Of the 26 patients for whom data were available, 22 (85%) were anatomically or functionally asplenic. In 25 (96%) of the 26 patients, B. holmesii was the only organism isolated from blood samples, and 14 patients (54%) had B. holmesii recovered from > or =2 blood cultures. The clinical course of the infection was generally characterized by a nonspecific febrile illness. Twenty-one patients (81%) were treated with various antimicrobial agents, and 20 (77%) were admitted to the hospital. There were no deaths. Our findings support evidence that B. holmesii may be a true pathogen associated with bacteremia among asplenic patients.

Tetanus immunity and physician compliance with tetanus prophylaxis practices among emergency department patients presenting with wounds.

STUDY OBJECTIVE: We determine tetanus seroprotection rates and physician compliance with tetanus prophylaxis recommendations among patients presenting with wounds. METHODS: A prospective observational study of patients aged 18 years or older who presented to 5 university-affiliated emergency departments (EDs) because of wounds was conducted between March 1999 and August 2000. Serum antitoxin levels were measured by enzyme immunoassay with seroprotection defined as more than 0.15 IU/mL. Seroprotection rates, risk factors for lack of seroprotection, and rates of physician compliance with tetanus prophylaxis recommendations by the Advisory Committee on Immunization Practices were determined. RESULTS: The seroprotection rate among 1,988 patients was 90.2% (95% confidence interval 88.8% to 91.5%). Groups with significantly lower seroprotection rates were persons aged 70 years or older, 59.5% (risk ratio [RR] 5.2); immigrants from outside North America or Western Europe, 75.3% (RR 3.7); persons with a history of inadequate immunization, 86.3% (RR 2.9); and persons without education beyond grade school, 76.5% (RR 2.5). Despite a history of adequate immunization, 18% of immigrants lacked seroprotection. Overall, 60.9% of patients required tetanus immunization, of whom 57.6% did not receive indicated immunization. Among patients with tetanus-prone wounds, appropriate prophylaxis (ie, tetanus immunoglobulin and toxoid) was provided to none of 504 patients who gave a history of inadequate primary immunization (of whom 15.1% had nonprotective antibody titers) and to 218 (79%) of 276 patients who required only a toxoid booster. CONCLUSION: Although seroprotection rates are generally high in the United States, the risk of tetanus persists in the elderly, immigrants, and persons without education beyond grade school. There is substantial underimmunization in the ED (particularly with regard to use of tetanus immunoglobulin), leaving many patients, especially those from high-risk groups, unprotected. Better awareness of tetanus prophylaxis recommendations is necessary, and future tetanus prophylaxis recommendations may be more effective if they are also based on demographic risk factors.

Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B. mallei.

Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa. We sequenced the 1.5-kb 16S rRNA gene of 56 B. pseudomallei and 23 B. mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity. Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference. Nine 16S types were identified in B. pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp. Twenty-two of 23 B. mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11. This report provides a basis for rapidly identifying and differentiating B. pseudomallei and B. mallei by molecular methods.

Paracoccus yeeii sp. nov. (formerly CDC group EO-2), a novel bacterial species associated with human infection.

CDC eugonic oxidizer group 2 (EO-2) is a group of unclassified gram-negative bacterial strains isolated from various human sources. As determined by biochemical tests and analyses of fatty acid compositions, these organisms form a homogeneous group that appears to be distinct from but related to other Paracoccus species. Molecular studies were performed on a set of 13 EO-2 strains from various clinical sources and geographic locations in the United States and Canada to determine their relationship to the Paracoccus genus. Control strains were Paracoccus denitrificans ATCC 17741(T), P. versutus ATCC 25364(T), P. aminophilus ATCC 49673(T), P. solventivorans ATCC 700252(T), and Psychrobacter immobilis ATCC 43116(T), which are phenotypically similar to EO-2. Nearly complete (1,500-base) 16S rRNA gene sequencing of eight EO-2 strains showed a high level of sequence similarity (>99.3%) within the group, and a BLAST search of GenBank placed the EO-2 cluster in close proximity to Paracoccus species (95 to 97% similarity). DNA-DNA hybridization studies of 13 of the EO-2 strains showed all to be related at the species level, with >70% relatedness under stringent conditions and a divergence within the group of less than 2%. None of the Paracoccus control strains hybridized at >54% with any of the EO-2 strains. These results indicate that EO-2 represents a new Paracoccus species, the first isolated from human clinical specimens. A new species, Paracoccus yeeii, is proposed for the EO-2 strains. The type strain of P. yeeii is CDCG1212 (ATCC BAA-599 and CCUG 46822), isolated in Pennsylvania from dialysate of a 77-year-old male with peritonitis.

Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis.

Four rapid tests for the serologic diagnosis of leptospirosis were evaluated, and the performance of each was compared with that of the current standard, the microscopic agglutination test (MAT). The four rapid tests were a microplate immunoglobulin M (IgM)-enzyme-linked immunosorbent assay (ELISA), an indirect hemagglutination assay (IHA), an IgM dipstick assay (LDS), and an IgM dot-ELISA dipstick test (DST). A panel of 276 sera from 133 cases of leptospirosis from four different geographic locations was tested as well as 642 sera from normal individuals or individuals with other infectious or autoimmune diseases. Acute-phase sera from cases (n = 148) were collected or=15 days after onset (median = 29.1). By a traditional method (two-by-two contingency table), the sensitivities for detection of leptospirosis cases were 93.2% by LDS, 92.5% by DST, 86.5% by ELISA, and 79.0% by IHA. Specificity was 98.8% by DST, 97% by ELISA and MAT, 95.8% by IHA, and 89.6% by LDS. With a latent class analysis (LCA) model that included all the rapid tests and the clinical case definition, sensitivity was 95.5% by DST, 94.5% by LDS, 89.9% by ELISA, and 81.1% by IHA. The sensitivity and specificity estimated by the traditional methods were quite close to the LCA estimates. However, LCA allowed estimation of the sensitivity of the MAT (98.2%), which traditional methods do not allow. For acute-phase sera, sensitivity was 52.7% by LDS, 50.0% by DST, 48.7% by MAT and ELISA, and 38.5% by IHA. The sensitivity for convalescent-phase sera was 93.8% by MAT, 84.4% by DST, 83.6% by LDS, 75.0% by ELISA, and 67.2% by IHA. A good overall correlation with the MAT was obtained for each of the assays, with the highest concordance being with the DST (kappa value, 0.85; 95% confidence interval [CI], 0.8 to 0.90). The best correlation was between ELISA and DST (kappa value, 0.86; 95% CI, 0.81 to 0.91). False-positive LDS results were frequent (>or=20%) in sera from individuals with Epstein-Barr virus, human immunodeficiency virus, and periodontal disease and from healthy volunteers. The ease of use and significantly high sensitivity and specificity of DST and ELISA make these good choices for diagnostic testing.

A two-component direct fluorescent-antibody assay for rapid identification of Bacillus anthracis.

A two-component direct fluorescent-antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA) antigens, was evaluated and validated for rapid identification of B. anthracis. We analyzed 230 B. anthracis isolates; 228 and 229 were positive by CW-DFA and CAP-DFA assays, respectively. We also tested 56 non-B. anthracis strains; 10 B. cereus and 2 B. thuringiensis were positive by the CW-DFA assay, and 1 B. megaterium strain was positive by CAP-DFA. Analysis of the combined DFA results identified 227 of 230 B. anthracis isolates; all 56 strains of the other Bacillus spp. were negative. Both DFA assays tested positive on 14 of 26 aging clinical specimens from the 2001 anthrax outbreak investigation. The two-component DFA assay is a sensitive, specific, and rapid confirmatory test for B. anthracis in cultures and may be useful directly on clinical specimens.

Dysgonomonas mossii sp. nov., from human sources.

Phenotypic and phylogenetic studies were performed on seven unidentified gram-negative, facultatively anaerobic, coccobacillus-shaped organisms isolated from human clinical specimens. Comparative 16S rRNA gene sequencing demonstrated that four of the strains corresponded to Dysgonomonas capnocytophagoides whereas the remaining three isolates represent a new sub-line within the genus Dysgonomonas, displaying greater than 5% sequence divergence with Dysgonomonas capnocytophagoides and Dysgonomonas gadei. The three novel isolates were readily distinguished from D.capnocytophagoides and D. gadei by biochemical tests. The DNA base composition of the novel species was consistent with its assignment to the genus Dysgonomonas. Based on phylogenetic and phenotypic evidence it is proposed that the unknown species, be classified as Dysgonomonas mossii sp. nov. The type strain of Dysgonomonas mossii is CCUG 43457T (= CIP 107079T).

Antimicrobial susceptibility testing of Bacillus anthracis: comparison of results obtained by using the National Committee for Clinical Laboratory Standards broth microdilution reference and Etest agar gradient diffusion methods.

We determined the patterns of antimicrobial susceptibility of 65 isolates of Bacillus anthracis (50 historical and 15 recent U.S. clinical isolates) to nine antimicrobial agents using the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution reference method. The results for the 50 historical B. anthracis isolates obtained by the broth microdilution method were compared to those generated by the Etest agar gradient diffusion method. One isolate of B. anthracis was beta-lactamase positive and resistant to penicillin (MIC, 128 microg/ml); a second isolate, which was beta-lactamase negative, was borderline penicillin resistant, with the penicillin MICs for the isolate varying from 0.12 to 0.25 microg/ml; and the remainder of the isolates were beta-lactamase negative and penicillin susceptible (MICs, or=16 microg/ml). All B. anthracis isolates were susceptible to chloramphenicol (MICs,

Clinical significance and epidemiology of NO-1, an unusual bacterium associated with dog and cat bites.

From 1974 to 1998, 22 isolates of an unusual bacterium, designated as CDC nonoxidizer 1 group (NO-1), were sent to the Centers for Disease Control and Prevention for identification. The organism's phenotypic characteristics were similar to asaccharolytic strains of Acinetobacter, but differed in their cellular morphology and cellular fatty acid profile. We report here on NO-1's clinical and epidemiologic significance. In all cases, isolates were recovered from an animal bite wound; 17 (77%) were isolated from a dog bite wound, 4 (18%) from a cat bite wound, and one (5%) from an unspecified animal bite. Clinical data were retrieved and reviewed for 12 (55%) of the 22 bite victims. None of the patients had pre-existing conditions associated with immunosuppression. Seven (58%) patients were hospitalized for a median stay of 4 days (range, 2 to 11 days). The median time between bite to the worsening of symptoms was 17.5 hours (range, 3 to 78 hours). All patients recovered following antibiotic treatment.

Changes in predominance and diversity of genomic subtypes of Bordetella pertussis isolated in the United States, 1935 to 1999.

Pulsed-field gel electrophoresis (PFGE) of Bordetella pertussis chromosomal DNA fragments generated by XbaI restriction has been used to subtype isolates for epidemiologic studies. To better understand the natural history of pertussis, we determined the PFGE profiles of 1,333 strains isolated in the United States from 1935 to 1999. Results showed a shift in prevalent profiles from the earliest to the latest study periods. In addition, genetic diversity decreased over time, and prevalent profiles were more highly related to each other than to less common profiles. These results provide the foundation for investigating the impact of prevention strategies, including the use of the acellular vaccines, on the currently circulating B. pertussis population.