Development and evaluation of a broad-range PCR-ELISA assay with Borrelia burgdorferi and Streptococcus pneumoniae as model organisms for reactive arthritis and bacterial meningitis.

We have developed an assay based on a 16S rDNA broad-range amplification system followed by species-specific detection with a commercially available PCR-ELISA kit. B. burgdorferi and S. pneumoniae were used as model systems for arthritis and meningitis, respectively. The sensitivity of the B. burgdorferi assay was comparable to that of a species-specific PCR, whereas for S. pneumoniae the detection limit was one to three organisms as determined by plate counts. To specifically differentiate two species, two discontinuously located nucleotide differences in the region complementary to the capture probe are required during the detection step with the PCR-ELISA kit. A preliminary clinical evaluation was performed with eight specimens (joint and cerebrospinal fluids) previously shown to contain B. burgdorferi DNA. Except for one sample which was positive by the broad-range PCR-ELISA system only, the results were in agreement with those obtained by B. burgdorferi species-specific PCR. None of the 23 control samples were positive by either method. Thus, broad-range amplification in combination with the PCR-ELISA kit promises to be a sensitive and specific format for the detection of agents causing reactive arthritis, meningitis or other diseases associated with a limited number of different bacteria.

Aeromonas popoffii sp. nov., a mesophilic bacterium isolated from drinking water production plants and reservoirs.

We examined the taxonomic position of seven Aeromonas isolates, recovered from Flemish and Scottish drinking water production plants and reservoirs, which were previously recognized by numerical analysis of genomic AFLP fingerprints as members of an unknown Aeromonas taxon that most closely resembled the species Aeromonas bestiarum (DNA hybridization group [HG] 2). The new phenotypic and DNA-DNA hybridization data obtained in this study show that the A. bestiarum-like strains constitute a separate Aeromonas species, for which the name Aeromonas popoffii sp. nov. is being proposed. The new species exhibited an internal DNA relatedness ranging from 79 to 100% and was 22 to 63% related to the type or reference strains of other Aeromonas spp. The highest DNA binding values were determined with A. bestiarum (51 to 63%), followed by Aeromonas hydrophila sensu stricto (HG1; 50 to 60%) and Aeromonas salmonicida (HG3; 39 to 55%). Although fingerprints generated by ribotyping and cellular fatty acid analysis often were highly similar, minor differences between the respective fingerprints were of significance for the differentiation of A. popoffii from its closest taxonomic neighbors, HG1, HG2, and HG3. Phenotypically, all seven strains of A. popoffii were positive for acid and gas production from D-glucose and glycerol, growth in KCN broth, arginine dihydrolase, DNase, Voges-Proskauer reaction, and resistance to vibriostatic agent O/129 and ampicillin but displayed negative reactions for production of urease, tryptophan deaminase, ornithine decarboxylase, and lysine decarboxylase (LDC). None of the strains displayed strong hemolytic activity. The lack of D-sucrose fermentation and LDC production and the ability to utilize DL-lactate as the sole energy and carbon source were useful characteristics for the biochemical separation of A. popoffii from A. bestiarum. Other Aeromonas spp. could be differentiated phenotypically from the new species by at least two features. The chromosomal G+C content of A. popoffii ranges from 57.7 to 59.6 mol%. Strain LMG 17541 is proposed as the type strain.

[Effect of ciprofloxacin therapy in duration of bacterial excretion in acute salmonella gastroenteritis]. / Einfluss einer Ciprofloxacin-Therapie auf die Ausscheidedauer bei akuter Salmonellen-Gastroenteritis.

In view of the increasing incidence of non-typhoid salmonellosis, the effect of early treatment with ciprofloxacin on the permanent elimination of salmonella was evaluated. In a prospective study, 14 patients with non-typhoid salmonellosis were treated with 2 x 500 mg/d ciprofloxacin for 10 days within 5 days of onset of symptoms. Relapse occurred in 4/14 (29%) patients 2-3 weeks after termination of therapy. Using sero- and ribotyping, relapse was confirmed and reinfection ruled out in 4/4 patients. Furthermore, ribotyping suggested double infection in one patient. Development of resistance to ciprofloxacin was not observed. We conclude that the use of antimicrobial chemotherapy to treat non-institutional salmonellosis in immunocompetent patients cannot generally be recommended but must be considered carefully in each case. Indications for ciprofloxacin therapy in the treatment of non-typhoid salmonellosis are discussed.

Isolation of gram-positive rods that resemble but are clearly distinct from Actinomyces pyogenes from mixed wound infections.

Beginning in 1990, gram-positive rods resembling Actinomyces pyogenes were found with increasing frequency in mixed cultures from various infectious processes, most of them from patients with otitis, empyema, pilonidal cysts, perianal abscesses, and decubitus ulcers. Ribotyping and hybridization showed that these gram-positive rods could be divided into five groups not related to known Actinomyces species. Biochemical markers for reliable differentiation into these groups, however, could not be found. Therefore, naming new species is not warranted unless parameters are discovered that allow identification without DNA hybridization. These gram-positive rods have been isolated only in mixed cultures with anaerobes, Staphylococcus aureus, Streptococcus "milleri," enterococci, and gram-negative rods. Their exact role in these possibly synergistic infections needs further investigation.

Development of resistance to quinolones in five patients with campylobacteriosis treated with norfloxacin or ciprofloxacin.

Development of resistance to nalidixic acid, norfloxacin and ciprofloxacin was observed in five patients with Campylobacter jejuni or Campylobacter coli infection. From all these patients nalidixic acid- and quinolone-susceptible strains were isolated initially, whereas after therapy with norfloxacin or ciprofloxacin strains resistant to these antibiotics were found. Campylobacter strains from the same patient always belonged to the same species and, with the exception of one case, showed identical rRNA gene restriction (rDNA) patterns. This indicates that double-infection with a susceptible and a resistant strain was not responsible for the phenomenon but rather that the infecting strain rapidly developed resistance following treatment.

Methods for the identification of DNA hybridization groups in the genus Aeromonas.

Among the 269 substrates tested in assimilation tests we found some that may help in the identification of DNA hybridization groups in the genus Aeromonas. In addition, isoenzyme analysis and ribotyping seem to be accurate although not routine procedures that allow discrimination between genetic species.

In vitro activities of fleroxacin, cefetamet, ciprofloxacin, ceftriaxone, trimethoprim-sulfamethoxazole, and amoxicillin-clavulanic acid against rare members of the family Enterobacteriaceae primarily of human (clinical) origin.

Fleroxacin and cefetamet were evaluated in vitro against 38 infrequently encountered species (250 strains) of the family Enterobacteriaceae and compared with four established compounds. For all the strains tested, the fleroxacin MIC was less than or equal to 0.5 mg/liter, and for 98% of strains the cefetamet MIC was less than or equal to 8 mg/liter; even though the two new compounds did not quite reach the activities (on a weight-by-weight basis) of ciprofloxacin and ceftriaxone, respectively, they nonetheless clearly surpassed trimethoprim-sulfamethaxazole and amoxicillin-clavulanic acid. The very potent new oral compounds tested in this study appear to be promising for the treatment of clinically relevant infections due to uncommon species of Enterobacteriaceae.

Biochemical identification of Aeromonas genospecies isolated from humans.

One hundred phenotypic characteristics were determined for 138 clinical and environmental Aeromonas strains. Cluster analysis revealed three major phenons equivalent to the A. hydrophila, A. caviae, and A. sobria groups, each of which contained more than one genospecies and more than one named species. An excellent correlation was found between phenotypic identification and classification based on DNA relatedness. DNA hybridization groups within each of the phenotypic groups were also separable by using a few biochemical characteristics. Key tests were production of acid from or growth on D-sorbitol (which separated DNA hybridization group 3 from groups 1 and 2 within the A. hydrophila phenogroup), growth on citrate (which essentially separated DNA hybridization group 4 from groups 5A and 5B within the A. caviae phenogroup), and growth on DL-lactate (which separated DNA hybridization group 1 from groups 2 and 3 within the A. hydrophila phenogroup as well as group 5A from groups 4 and 5B within the A. caviae phenogroup). All except one strain in the A. sobria phenogroup belonged to DNA hybridization group 8. DNA hybridization groups were not equally distributed among clinical and environmental isolates, suggesting that strains of certain DNA hybridization groups might be less virulent than others.

Automated analysis of 35-S-methionine labeled proteins by SDS-PAGE as a typing method in a suspected cluster of Serratia marcescens.

During a nine-month period in 1986, we observed five ornithine decarboxylase-negative isolates of Serratia marcescens from four different patients. All isolates were identical in more than 50 biochemical parameters. Four isolates from three hospitalized patients were essentially identical in their susceptibility patterns to 12 antimicrobial agents. Analysis of 35-S-methionine labeled whole cell proteins by SDS-PAGE with the AMBIS system (Automated Microbiology System Inc., San Diego, CA) suggested that only three of these isolates were identical while the fourth hospital strain was more closely related to the isolate of a patient with no contact to the hospital. These results were confirmed by bacteriocin- and serotyping. We conclude that analysis of these protein patterns--which does not require special reagents - was an adequate method for typing S. marcescens.