Diagnosis of gonorrhoea using a genetic transformation test at a venereal disease clinic in Thailand.

A genetic transformation test (GTT), a technique in which gonococcal DNA is detected in clinical specimens, was used to search for Neisseria gonorrhoeae infections in 37 men and 159 women at the Venereal Disease clinic in Cholburi, Thailand. Swabs were collected in duplicate from cervical specimens from 159 women and from urethral specimens from 37 men. One of each specimen was cultured on Thayer-Martin media while the other was mailed to the United States at room temperature for the GTT which involved a delay of 10 to 14 days. With the urethral specimens N. gonorrhoeae was identified in 84% (31/37) of specimens and there was 100% concordance between the results of the GTT and culturing specimens directly on Thayer-Martin media. With cervical specimens N. gonorrhoeae was isolated from 26% (41/159) by the standard culture technique and 19% (13/159) by the GTT. Seventy-six percent of the culture positive specimens were positive with the GTT and two specimens from which N. gonorrhoeae were not isolated were positive in the GTT. The GTT technique enables physicians to send swab collected from patient with suspected gonorrhoea without any special transport media to a central laboratory for laboratory diagnosis of gonorrhoeal infections. This technique which uses reagents which are available in most bacteriology laboratories, should facilitate surveillance of gonorrhoea especially when specimens are collected in clinics where bacteriology laboratory facilities are not available.

Diagnosis of gonorrhea using a genetic transformation test on mailed clinical specimens.

The genetic transformation test (GTT), a technique used for the detection of gonococcal DNA in clinical specimens, was compared with culture testing for the diagnosis of gonorrhea. At the De Kalb County Venereal Disease Clinic, Decatur, Ga., 454 cervical and 160 rectal specimens from women and 191 urethral specimens from men were collected in duplicate. One of each of the two specimens from each anatomic site was immediately plated on Martin-Lewis medium and incubated; the other specimen was mailed to Philadelphia for a GTT. Using culture results as a standard, the GTT had a specificity of 98.1% although some "false-positive" GTT results were probably a reflection of false-negative culture results. The sensitivity of the GTT was greater than 96% except when specimens were collected with Culturettes (Marion Laboratories, Kansas City, Mo.). In situations where Gram staining is not appropriate or where on-site culture facilities are not available, GTT may be the method of choice for the diagnosis of gonorrhea.

Laboratory diagnosis of gonorrhea by a simple transformation test with a temperature-sensitive mutant of Neisseria gonorrhoeae.

A temperature-sensitive mutant of Neisseria gonorrhoeae strain tsA-1 was used in a transformation test for the laboratory diagnosis of gonorrhea. This transformation test (C test) is based on a spotting of a DNA lysate, obtained through simple base-acid extraction of a cervical-swab specimen, onto a lawn of tsA-1, which is then incubated for one to two days at 37 C. Of 1,053 cervical-swab specimens tested, 52 contained the gonococcal DNA necessary for restoring the ability of tsA-1 to grow well at 37 C; 49 of the 52 specimens were identified as N. gonorrhoeae by routine diagnostic laboratory procedures. The time required for the C test is two to three days, which is shorter than that required for routine diagnostic laboratory tests, and the C test involves a minimum of common laboratory supplies and expertise. The discrepancies between results of the C test and routine procedures are explained as follows. In one case an isolate suspected to be N. gonorrhoeae was nonreactive in the sugar fermentation test, and in two cases the agar plates were overgrown with yeasts; neither situation affected the C test.

Polygenes and modifier genes for tetracycline and penicillin resistance in Neisseria gonorrhoeae.

The genetic basis for spontaneous resistance to tetracyline (Tet) and penicillin (Pen) in Neisseria gonorrhoeae was investigated. Tet and pen are polygenes which confer small but distinct levels of resistance to Tet and Pen, respectively. Mtr is a multiple-drug resistance polygene which increases resistance to Tet and Pen (as well as to other unrelated antibiotics). Tem is a modifier gene affecting resistance toTet and Pen. Pem is a modifier gene for Pen resistance. The following gene combinations code for resistance to five antibiotics: tet, mtr and tem for Tet; pen, mtr, pem and tem for Pen; tet, tem and mtr for doxycycline; pen and pem for ampicillin; pen, pem and mtr for nafcillin.

Temperature-sensitive mutants of Neisseria gonorrhoeae.

Nine temperature-sensitive (Ts) mutants of Neisseria gonorrhoeae strain 49191 were isolated. They were proven to be different from each other by results of transformation experiments. None of the Ts mutations appeared to be linked to antibiotic resistance genes from strain 24392. However, Ts-9 demonstrated 8% linkage with a nalidixic acid resistance marker from strain RW-2.

Genetic analysis of drug resistance in Neisseria gonorrhoeae: production of increased resistance by the combination of two antibiotic resistance loci.

The studies reported here demonstrate that increased resistance of Neisseria gonorrhoeae to penicillin, tetracycline, and chloramphenicol results from the combined effect of two resistance loci. As shown by experiments with deoxyribonucleic acid from transformants carrying only a single resistance locus, transformants with an incresed level of resistance to penicillin result from the combination of a penicillin-specific locus, pen, and a multiple resistance locus, mtr. Similarly, transformants with an increased level of resistance to tetracycline result from the combination of mtr and a tetracycline-specific locus, tet. Transformants with an increased level of resistance to chloramphenicol result from the combination of mtr and a chloramphenicol-specific locus, cml. Deoxyribonucleic acid dilution experiments established that only a single dose of each of the two required resistance loci is necessary to give higher-level resistance. Higher-level-resistant transformants were not obtained when a double dose of one resistance locus or a combination of loci pairs other than mtr and pen, mtr and tet, or mtr and cml was introduced into a recipient. Combinations of the mtr and tet genes resulted in increased resistance to semisynthetic tetracyclines. The presence of the mtr and pen genes resulted in increased resistance to penicillinase-stable penicillins.

Genetic analysis of drug resistance in Neisseria gonorrhoeae: identification and linkage relationships of loci controlling drug resistance.

The genetic basis of multiple drug resistance of Neisseria gonorrhoeae was investigated by the technique of transformation. Six different genetic loci were characterized by the type and amount of antibiotic resistance they controlled, and also by the degree of linkage to other resistance markers. A streptomycin resistance locus is linked to separate loci determining resistance to tetracycline, chloramphenicol, and erythromycin. A multiple resistance locus was identified. This genetic locus determines resistance to a variety of antibacterial agents. Lastly, a locus determining resistance to the penicillins was found which is unlinked to any other resistance locus.

Multiple antibiotic resistance in Neisseria gonorrhoeae.

The sensitivities of gonococcal isolates to six antibiotics were determined for gonococci isolated in Philadelphia in 1972. The degree of association between susceptibilities to any two antibiotics was determined (coefficient of correlation). The correlation between penicillin and tetracycline (r = 0.75) was almost as good as that between two penicillins, penicillin G and ampicillin (r = 0.85), but the difference was statistically significant. The lowest correlation found was between erythromycin and chloramphenicol (r = 0.62), two antibiotics seldom used in gonorrhea therapy. In addition, gonococci most resistant to one antibiotic were the most likely to be multiply resistant. This was found with respect to penicillin, tetracycline, erythromycin, and chloramphenicol. Approximately 40% of gonococci classified as "most resistant" (exceeding the resistance of 75% of all isolates) to one antibiotic were also "most resistant" to three others. Finally, multiply resistant mutants were isolated by selection for resistance to either penicillin or tetracycline. These results provide evidence for the existence of a common mechanism for multiple antibiotic resistance in the gonococcus.

Antibiotic disk susceptibility tests with Neisseria gonorrhoeae.

We have used a standardized disk diffusion procedure to test the susceptibility of 892 isolates of Neisseria gonorrhoeae to six antibiotics. The disk diffusion test described is a modification of the disk test of Bauer, Kirby, Sherris, and Turck, which is widely used for susceptibility testing of fast-growing, aerobic pathogens. With all six antibiotics (penicillin, ampicillin, tetracycline, erythromycin, chloramphenicol, and cephaloridine), disk test results were found to be reproducible and correlated well with the minimal inhibitory concentration as determined by an agar dilution procedure. The coefficients of correlation ranged from -0.79 with cephaloridine to -0.93 with penicillin. The technique described for disk susceptibility testing of gonococci can be used both for research and clinical purposes.

Technique for screening 2n combinations of nutritional requirements.

An auxanographic technique is described, which makes it possible to screen 2(n) combinations of nutrients on relatively few agar plates.