Pasteurella multocida subsp. multocida and P. multocida subsp. septica differentiation by PCR fingerprinting and alpha-glucosidase activity.

Pasteurella multocida is composed of three subspecies that are often differentiated by fermentation of sorbitol and dulcitol. We studied 35 dulcitol-negative P. multocida isolates from infected dog and cat bite wounds, 16 of which yielded weak and/or conflicting fermentation reactions in Andrades sorbitol, thus making it difficult to distinguish between the two dulcitol-negative subspecies of P. multocida, i.e., P. multocida subsp. multocida and P. multocida subsp. septica. All isolates and two control strains were further analyzed using a PCR fingerprinting technique with a single primer (M13 core) and assessed for alpha-glucosidase (alpha-Glu) activity. Although the PCR fingerprint patterns and alpha-Glu activity did not correlate well with the sorbitol fermentation reactions, they did correlate well with each other. All strains identified as P. multocida subsp. septica were positive for alpha-Glu activity and exhibited the group I PCR fingerprint profile. All strains categorized as P. multocida subsp. multocida displayed either the group II or group III PCR fingerprint profile; 9 of 11 of these isolates were alpha-Glu negative. These data suggest that both PCR fingerprinting and alpha-Glu activity provide reliable means for differentiating P. multocida subsp. multocida from P. multocida subsp. septica, particularly in strains that produce weak and/or discrepant sorbitol fermentation reactions.

PCR fingerprinting analysis for differentiation of Streptococcus pneumoniae reinfection versus relapse.

Polymerase chain reaction fingerprint profiles of isolates obtained during an episode of pneumococcal pneumonia with bacteremia differed significantly from profiles of isolates obtained from the same patient during a subsequent episode of pneumococcal meningitis with bacteremia. Polymerase chain reaction fingerprinting provides a means of differentiating new infection from relapse, and may be a simple molecular tool for comparison of Streptococcus pneumoniae isolates.

Characterization of Bacteroides forsythus strains from cat and dog bite wounds in humans and comparison with monkey and human oral strains.

Bacteroides forsythus strains recovered from cat and dog bite wound infections in humans (n = 3), monkey oral strains (n = 3), and the human oral ATCC 43037 type strain were characterized by using phenotypic characteristics, enzymatic tests, whole cell fatty acid analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, PCR fingerprinting, and 16S rDNA (genes coding for rRNA) sequencing. All three bite wound isolates grew on brucella agar supplemented with 5% sheep blood, vitamin K1, and hemin. These strains, unlike the ATCC strain and previously described monkey oral and human clinical strains, did not require N-acetylmuramic acid supplementation for growth as pure cultures. However, their phenotypic characteristics, except for catalase production, were similar to those of previously identified strains. PCR fingerprinting analysis showed differences in band patterns from the ATCC strain. Also, SDS-PAGE and whole cell fatty acid analysis indicated that the dog and cat bite wound strains were similar but not identical to the human B. forsythus ATCC 43037 type strain and the monkey oral strains. The rDNA sequence analysis indicated that the three bite wound isolates had 99.93% homology with each other and 98.9 and 99.22% homology with the human ATCC 43037 and monkey oral strains, respectively. These results suggest that there are host-specific variations within each group.

Activities of HMR 3004 (RU 64004) and HMR 3647 (RU 66647) compared to those of erythromycin, azithromycin, clarithromycin, roxithromycin, and eight other antimicrobial agents against unusual aerobic and anaerobic human and animal bite pathogens isolated from skin and soft tissue infections in humans.

The activities of HMR 3004 and HMR 3647 and comparator agents, especially macrolides, were determined by the agar dilution method against 262 aerobic and 120 anaerobic strains isolated from skin and soft tissue infections associated with human and animal bite wounds. HMR 3004 and HMR 3647 were active against almost all aerobic and fastidious facultative isolates (MIC at which 90% of the isolates are inhibited [MIC90], < or = 0.5 and 1 microg/ml, respectively) and against all anaerobes [Bacteroides tectum, Porphyromonas macacae (salivosa), Prevotella heparinolytica, Porphyromonas sp., Prevotella sp., and peptostreptococci] at < or = 0.25 and < or = 0.5 microg/ml, respectively, except Fusobacterium nucleatum (HMR 3004, MIC90 = 16 microg/ml; HMR 3647, MIC90 = 8 microg/ml) and other Fusobacterium species (MIC90, 1 and 2 microg/ml, respectively). In general, HMR 3004 and HMR 3647 were more active than any of the macrolides tested. Azithromycin was more active than clarithromycin against all Pasteurella species, including Pasteurella multocida subsp. multocida, Eikenella corrodens, and Fusobacterium species, while clarithromycin was more active than azithromycin against Corynebacterium species, Weeksella zoohelcum, B. tectum, and P. heparinolytica.

Trovafloxacin compared with levofloxacin, ofloxacin, ciprofloxacin, azithromycin and clarithromycin against unusual aerobic and anaerobic human and animal bite-wound pathogens.

The activity of trovafloxacin and five other oral agents against 250 aerobic and 137 anaerobic strains isolated from human and animal bite wounds was determined by an agar dilution method. Trovafloxacin was active against all aerobic and fastidious facultative isolates at < or = 0.5 mg/L and all anaerobes at < or = 2 mg/L (Bacteroides tectum, Porphyromonas salivosa and Prevotella heparinolytica, < or = 0.25 mg/L; Porphyromonas spp., < or = 0.5 mg/L; Prevotella spp. and peptostreptococci, < or = 2.0 mg/L), except Fusobacterium nucleatum and other fusobacteria (MIC90 < or = 4 mg/L). Levofloxacin was generally one to two dilutions more active than ofloxacin, while ciprofloxacin was active against aerobes (MIC < or = 1 mg/L) but less active against anaerobic strains (MIC90 < or = 16 mg/L).

Evaluation of the RapID CB Plus system for identification of Corynebacterium species and other gram-positive rods.

Due to the difficulty of identifying Corynebacterium spp. with standard methods, we compared them with the RapID CB Plus system (Remel, Lenexa, Kans. [formerly Innovative Diagnostic Systems, Norcross, Ga.]), which consists of 4 carbohydrate and 14 preformed enzyme tests, for the identification of 98 clinical isolates of Corynebacterium sp., other coryneforms, Listeria monocytogenes, and 17 ATCC strains. Forty (95%) of 42 strains of Corynebacterium spp. were accurately identified to the species level by the RapID CB Plus system, and two additional strains of C. striatum were identified with one additional conventional test for lipid requirement. Twenty-seven (75%) of the 36 coryneform strains tested were identified correctly to the species level. However, three of four strains of Brevibacterium sp. and all seven of the L. monocytogenes strains were identified to the genus level only. Actinomyces strains had variable results, and the one strain of Arcanobacterium haemolyticum tested was not identified. Overall, the RapID CB Plus system compared favorably with the conventional methods, was easy to inoculate and interpret, and is promising as a new method for identification of gram-positive bacilli.

Bilophila wadsworthia clinical isolates compared by polymerase chain reaction fingerprinting.

Bilophila wadsworthia isolates recovered from a right-ear cholesteatoma and brain abscess of the same patient were analyzed by means of polymerase chain reaction (PCR) fingerprinting with single primers (T3B and M13 core) to ascertain if they originated from the same clone. Their PCR fingerprint profiles were compared with those of three additional B. wadsworthia clinical isolates and the type strain (ATCC 49260). The two isolates from the same patient produced PCR fingerprint profiles identical to each other, regardless of which primer was used. All isolates' PCR fingerprint profiles, with use of either the T3B or M13 core primer, shared some major and minor bands. However, differences in additional major and minor bands distinguished each of the additional isolates, suggesting that there are different subgroups of B. wadsworthia.

Use of polymerase chain reaction fingerprinting to compare clinical isolates of Bacteroides fragilis and Bacteroides thetaiotaomicron from Germany and the United States.

Accurate identification of Bacteroides species is often problematic. Therefore, we used a polymerase chain reaction (PCR) fingerprinting technique with either a single nonspecific primer derived from tDNA intergenic spacer or a single primer that anneals to mini- and microsatellite DNA sequences to compare 34 clinical isolates of B. fragilis and 21 clinical isolates of B. thetaiotaomicron from Southern California with 32 clinical isolates of B. fragilis and 10 isolates of B. thetaiotaomicron from Germany. All German B. fragilis isolates (32 of 32) formed one PCR fingerprint group that matched the PCR profile of the B, fragilis reference strain ATCC (American Type Culture Collection) 25285, representative of DNA homology group I. In contrast, the isolates from Southern California formed two PCR fingerprint groups. Although most of these strains (29 of 34) also matched B. fragilis ATCC 25285, some strains (4 of 34) matched the DNA homology group II reference strain VPI (Virginia Polytechnic Institute) 2393. One of the 34 strains showed a unique profile. German B. thetaiotaomicron strains (10 of 10) formed one PCR fingerprint group, matching the reference strain B. thetaiotaomicron ATCC 29742, whereas the B. thetaiotaomicron isolates from Southern California showed heterogenous profiles.

Growth characteristics and a novel method for identification (the WEE-TAB system) of Porphyromonas species isolated from infected dog and cat bite wounds in humans.

Thirty-nine clinical isolates of Porphyromonas species recovered from infected cat and dog bite wounds in humans and eight American Type Culture Collection and National Collection of Type Cultures type strains were characterized by using the API ZYM system, the RapID ANA II system, and conventional biochemical methods. Growth characteristics on various agar media were compared. All strains grew on brucella blood agar supplemented with vitamin K1 and hemin and on brucella laked blood agar supplemented with vitamin K1 and hemin. In contrast, only 34% of strains grew on unsupplemented brucella blood agar, 62% grew on Columbia blood agar, and 70% grew on tryptic soy blood agar (the last three media did not contain vitamin K1 or hemin). The ability of the single-tube, triple-substrate WEE-TAB system to detect the preformed enzymes N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase, beta-D-galactosidase, alpha-fucosidase, trypsin-like activity, and chymotrypsin was evaluated. The WEE-TAB test results were easy to interpret; the WEE-TAB tests were more sensitive than the comparable tests with the API ZYM and RapID ANA II systems for the detection of alpha-D-galactosidase, beta-D-galactosidase, trypsin, and chymotrypsin, and the WEE-TAB tests accurately identified Porphyromonas species.

In vitro activity of Bay 12-8039, a new 8-methoxyquinolone, compared to the activities of 11 other oral antimicrobial agents against 390 aerobic and anaerobic bacteria isolated from human and animal bite wound skin and soft tissue infections in humans.

The in vitro activity of Bay 12-8039, a new oral 8-methoxyquinolone, was compared to the activities of 11 other oral antimicrobial agents (ciprofloxacin, levofloxacin, ofloxacin, sparfloxacin, azithromycin, clarithromycin, amoxicillin clavulanate, penicillin, cefuroxime, cefpodoxime, and doxycycline) against 250 aerobic and 140 anaerobic bacteria recently isolated from animal and human bite wound infections. Bay 12-8039 was active against all aerobic isolates, both gram-positive and gram-negative isolates, at < or = 1.0 microg/ml (MICs at which 90% of isolates are inhibited [MIC90s < or = 0.25 microg/ml) and was active against most anaerobes at < or = 0.5 microg/ml; the exceptions were Fusobacterium nucleatum and other Fusobacterium species (MIC90s, > or = 4.0 microg/ml) and one strain of Prevotella loeschii (MICs, 2.0 microg/ml). In comparison, the other quinolones tested had similar in vitro activities against the aerobic strains but were less active against the anaerobes, including peptostreptococci, Porphyromonas species, and Prevotella species. The fusobacteria were relatively resistant to all the antimicrobial agents tested except penicillin G (one penicillinase-producing strain of F. nucleatum was found) and amoxicillin clavulanate.

Comparative in vitro activities of DU-6859a, levofloxacin, ofloxacin, sparfloxacin, and ciprofloxacin against 387 aerobic and anaerobic bite wound isolates.

The activities of DU-6859a, levofloxacin, ofloxacin, sparfloxacin, and ciprofloxacin against bite wound isolates were determined by the agar dilution method. DU-6859a was the most active compound (MICs, < or = 0.125 microg/ml) against all Pasteurella species, Staphylococcus aureus, and streptococci; anaerobes were susceptible to < or = 0.5 microg/ml, except fusobacteria, which were susceptible to < or = 2 microg/ml. Against aerobes, levofloxacin was more active than ofloxacin (MIC at which 90% of isolates are inhibited [MIC90], < or = 1.0 microg/ml for both) and sparfloxacin and ciprofloxacin were also active (MIC90s, < or = 0.25 and < 1 microg/ml, respectively).

Characterization of saccharolytic Bacteroides and Prevotella isolates from infected dog and cat bite wounds in humans.

Saccharolytic, nonpigmented, anaerobic gram-negative rods isolated from infected dog and cat bite wounds in humans have been poorly characterized, and most are not included in the databases of kits used for anaerobic identification; thus, they are problematic for clinical laboratories to identify. Fifty strains isolated from such wounds were characterized with commercial kits for preformed-enzyme detection, carbohydrate fermentation, and other biochemical tests. PCR fingerprinting was performed on these strains to further characterize subgroups within these species. Bacteroides tectum is a frequent isolate in bite wounds and resembles Prevotella bivia in colony morphology and saccharolytic activity, except that it grows in 20% bile and hydrolyzes esculin. Profile numbers generated by various kits associate B. tectum with P. bivia, Prevotella oralis group, or Prevotella melaninogenica. PCR fingerprinting identified at least four subgroups and confirmed the heterogeneous nature of this species. Prevotella heparinolytica was also frequently isolated from these bite wounds. It produces indole and generates a profile number in preformed-enzyme kits that is usually associated with Bacteroides uniformis. However, it is bile sensitive and quite distinct from the Bacteroides fragilis group of anaerobes. The PCR fingerprint profiles generated by strains of P. heparinolytica were very similar to that of the type strain and to each other. Prevotella zoogleoformans, occasionally isolated from dog and cat bite wounds in humans, resembles P. heparinolytica except for a negative indole test. Clinical laboratories should be aware of the characteristics of these animal species when identifying isolates from animal bite wounds in humans.

Characterization of indole-negative Bacteroides fragilis group species with use of polymerase chain reaction fingerprinting and resistance profiles.

Biochemical tests alone do not adequately differentiate the various Bacteroides species, groups, and antimicrobial-resistant variants. Consequently, we used a polymerase chain reaction (PCR) fingerprinting technique, with either a single nonspecific primer derived from the t-DNA intergenic spacer region (T3B) or a single primer that anneals to minisatellite DNA sequences (M13 core), to identify and characterize 58 clinical isolates of Bacteroides fragilis group species (B. fragilis, B. distasonis, and B. caccae). In addition to species- and subspecies-specific differences, 4 strains of B. fragilis, 1 of B. distasonis, and 3 of B. caccae that showed increased resistance to imipenem, ampicillin, and ampicillin/sulbactam also produced unique PCR fingerprint profiles. Analysis by the clinical source of isolation (i.e. blood or intraabdominal, skin, or soft-tissue infection) indicated that no particular PCR fingerprint type was associated with greater pathogenicity of any individual clinical source. The PCR fingerprinting technique proves to be a useful tool for species identification and taxonomic studies, as well as for epidemiological studies of Bacteroides species.

Frequency of isolation of Porphyromonas species from infected dog and cat bite wounds in humans and their characterization by biochemical tests and arbitrarily primed-polymerase chain reaction fingerprinting.

We isolated 40 strains of Porphyromonas (formerly Bacteroides) species from 29 of 102 cat and dog bite wounds in humans. P. salivosa, P. gingivalis, and P. canoris were the most frequent isolates. A comparison of the RapID ANA II system (Innovative Diagnostic Systems, Norcross, GA), An-IDENT panels (bioMérieux, St. Louis), and API ZYM strips (bioMérieux) showed that the latter kit best characterized these isolates because it included tests for trypsin and chymotrypsin activity; however, the tests for glycosidase activity in this kit were less sensitive than were those in the other kits. None of the biochemical systems was able to identify all species. Arbitrarily primed-polymerase chain reaction fingerprinting with a nonspecific single primer, T3B, yielded distinct profiles for type strains and for the clinical isolates, suggesting that some of the isolates represented previously undescribed species. Growth of these species took > or = 5 days; therefore, laboratories should incubate anaerobic plates from bite wound cultures for > or = 7 days to assure isolation of these common pathogens.

Comparison of Etest to broth microdilution method for testing Streptococcus pneumoniae susceptibility to levofloxacin and three macrolides.

When the Etest was compared to broth microdilution for susceptibility testing of Streptococcus pneumoniae, levofloxacin, erythromycin, and penicillin results correlated for both methods; azithromycin and clarithromycin showed discrepancies of > or = 2 dilutions for 95.8% and 31.5% of the isolates, respectively. Levofloxacin was active against 141 of 142 isolates (< or = 2.0 micrograms/ml), making it a potentially useful new fluoroquinolone.

Clinical importance of Bilophila wadsworthia.

Bilophila wadsworthia is an anaerobic, gram-negative, asaccharolytic, urease-positive, bile-resistant, catalase-positive bacillus, originally recovered from infections in patients with gangrenous and perforated appendicitis. Additional isolations from clinical specimens, including pleural fluid, joint fluid, blood and pus from a scrotal abscess, mandibular osteomyelitis and axillary hidradenitis suppurativa are described here. Bilophila is found as normal flora in feces and, occasionally, in saliva and in the vagina. Isolates from humans are usually beta-lactamase positive and therefore resistant to certain beta-lactam antibiotics. Two percent of strains are also resistant to clindamycin.

Human IgG anti-F(ab')2 antibodies possess rheumatoid factor activity.

Individual preparations of affinity purified anti-F(ab')2 antibodies and anti-Fc antibodies isolated from the sera of patients with rheumatoid arthritis (RA), were examined for reactivity with the Fab and Fc fragments of human IgG. Western blot assays demonstrated specific interaction of affinity-purified anti-Fab antibodies with both Fab and Fc molecules. Approximately one-half of the anti-Fab antibody preparations studied contained IgG antibodies reactive with Fab and Fc fragments in ELISA, suggesting the existence of naturally occurring epibody-like autoantibodies in these patients. Thirteen of 14 affinity-purified anti-Fc antibody preparations contained IgG cross-reactive with Fab molecules in ELISA. Double-adsorption assays on affinity columns demonstrated that a minimum of 14%, and possibly as much as 50%, of the IgG anti-Fab antibodies reacted with the Fc of IgG. Conversely, a minimum of 12%, and possibly as much as 70%, of the IgG anti-Fc antibodies reacted with IgG Fab molecules. Anti-Fab antibodies isolated from non-RA individuals also exhibited anti-Fc reactivity in ELISA, demonstrating the presence of these dual-reactive antibodies in other autoimmune and normal individuals. These studies establish the presence of naturally occurring IgG autoantibodies reactive with both the Fab and Fc fragments of human IgG. Their existence emphasizes the potential of anti-immunoglobulin antibodies to recognize a multiplicity of antigens, possibly including other members of the immunoglobulin supergene family.

Recognition by anti-Fab antibodies in rheumatoid arthritis of structure(s) widely distributed on human Fab molecules.

Anti-Fab antibodies (aFABA) of restricted clonality and acidic spectrotypes were isolated from the sera of patients with rheumatoid arthritis (RA). These aFABA reacted with multiple populations of pooled human Fab molecules, which had been charge separated by chromatofocusing techniques (CF), indicating that the structures recognized by these aFABA were present on a polyclonal population of Fab molecules. The structures were also widely distributed among the Fab repertoires of normal individuals, as well as individual autologous and heterologous RA patients. Thus, the aFABA did not appear to recognize highly restricted epitope(s), i.e. a private idiotope, limited in its expression to RA individuals. The determinants of the Fab molecules recognized by affinity purified aFABA could be defined by linear and/or conformational structures, depending upon the individual from which the aFABA were isolated. Additionally, some of the affinity purified aFABA also reacted with Fc fragments, suggesting the presence of epibody-like autoantibodies in this population. Lastly, size analysis of the circulating IgG4 aFABA complexes indicated that these autoantibodies were not complexed with intact IgG, but rather with a molecule of 40-60 kDa, further suggesting the potential for these autoantibodies to react with multiple antigens.