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A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.

This study evaluates a cryptic plasmid-derived DNA probe in a dot-blot hybridization assay of 4-h duration, using both known bacterial isolates and clinical specimens. The probe, consisting of a 237 bp segment of the plasmid-encoded gene cppB, sequences of which are also found in the chromosome, was labelled with digoxigenin-11-dUTP. The sensitivity of the probe was approximately 25 pg of DNA or 500 cfu of Neisseria gonorrhoeae. A total of 170 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All N. gonorrhoeae strains, including three plasmid-free strains, hybridized with the probe. Among the heterologous bacterial cultures, only one strain of N. cinerea reacted with the probe when the cell concentration was 5 x 10(6) cfu. The probe was also evaluated in a clinical study. A total of 201 patients visiting the STD clinic at the University Hospital, University of Seville, participated in the study. The sensitivity of the assay was 95% while the specificity was 98%. Positive and negative predictive values were 97% and 98%, respectively. It appears that the plasmid-derived probe used in this study could serve as a useful tool in the rapid and specific detection of Neisseria gonorrhoeae in clinical specimens.

The diagnostic effectiveness of a new test for the detection of bacteriuria (Filtrachek-UTI) was compared with Gram stain and semiquantitative culture in 955 urine samples. 276 of these were from inpatients (108 males and 168 females), and 677 from outpatients (206 males and 471 females). Overall 229 positive cultures were obtained. Taking the sample culture as the standard, the statistical parameters obtained were very low, particularly the sensitivity and the negative predictive value (75-89% sensitivity and 80-91% NPV in inpatients, and 67-92% sensitivity and 79-98% NPV in outpatients). Other problems of the method are the subjective interpretation, choluria or filter obliteration by the urine, high cost and an approximate false negative rate of 16%.

The ecology of R plasmids was studied in the intestinal flora of 19 patients with salmonellosis without antibiotic treatment. The plasmids found in the Salmonella strains and the accompanying non-pathogenic Enterobacteriaceae were characterized in each patient. We determined the transferability by conjugation, the fi character and the incompatibility group and did enzyme restriction analysis of these plasmids. The results obtained showed that S. typhimurium is the species of this genus with the highest incidence of R plasmids, and Escherichia coli among the non-pathogenic Enterobacteriaceae. The plasmids found in Salmonella are different from the plasmids found in the other Enterobacteriaceae in fi character (50% fi+ in Salmonella and 5% in the other Enterobacteriaceae) and incompatibility group (33% belong to the FII group in Salmonella plasmids and none on the other Enterobacteriaceae), thereby expressing a different origin.

The in vitro activity of the new semisynthetic ureidopenicillins azlocillin (AZ) and mezlocillin (MZ), and of the new cephalosporin cefsulodin (CEF) were determined against 50 carbenicillin-sensitive (CARs) and 50 carbenicillin-resistant (CARr) P. aeruginosa clinical isolates. In the CARs group the most active antibiotics are AZ and CEF with MICs between 0.5 and 8 microgram/ml. Ticarcillin (TIC) and MZ showed more activity than CAR with MICs from 2 to 32 mu/ml. In this group there is a predominance of pyocine-type groups 1 and 3. In the CARr group, AZ is the most active antibiotic at low concentrations. At 64 microgram/ml of CEF, 72% of strains are inhibited as compared to 70% with AZ, 62% with MZ and 50% with TIC. In this group there is predominance of non-typeable strains. Interaction with mecillinam (MEC) and these antibiotics was studied on three different culture media (MH, NIH and DST). There were few cases of synergism with MEC and TIC combinations, mostly on DST medium. No appreciable synergism was found with other combinations of antibiotics.

A comparative study of a DNA probe for the detection of Neisseria gonorrhoeae with Gram stain and modified Thayer-Martin medium was performed. The probe was the 2.6 megadaltons (Mda) cryptic plasmid of N. gonorrhoeae, labeled with a nonradioactive system combining sulphonation of DNA with the antibody detection of sulphonated groups (Organics Chemiprobe). Overall 101 samples were evaluated: 39 urethral exudates, 3 rectal exudates, and 59 cervical exudates, from 42 males and 54 females in whom gonococcal infection was suspected. Simultaneously, Gram stain and culture of the several exudates were carried out. The in vitro sensitivity of the method was evaluated with different dilutions of N. gonorrhoeae, from 1 to 1000 cfu/ml, and a minimum of 50 cfu/ml were detected. In the detection of N. gonorrhoeae in clinical samples, there was a 100% sensitivity and a specificity of 83% (males) and 79% (females), with positive predictive value of 97% in males and 53% in females. The negative predictive value was 100% both for males and females.

The effects of subinhibitory concentrations (sub-MICs) of ampicillin on R plasmid transfer in Escherichia coli were studied. Each donor strain culture was separated into two parts; one was mixed with a recipient strain culture for mating, the other was treated with the sub-MIC of ampicillin and then mixed with the recipient strain culture. In both cases the R plasmid transfer frequency was determined at 30, 60, 90 and 120 min of mating. Results showed that there exists a general decrease in the transfer frequencies under sub-MIC treatment (two plasmids did not transfer at all). The proportion of aggregates and the number of cells that compose them were not affected by the sub-MIC of ampicillin. Our study supports the idea that the changes induced in E. coli by sub-MICs of ampicillin did not affect the function of the surface structures responsible for aggregation but did affect the proteins implicated in DNA transfer, situated on the cell surface.