Steven R. Johnson

High-level tetracycline resistance in Neisseria gonorrhoeae is result of acquisition of streptococcal tetM determinant.

Recently, strains of Neisseria gonorrhoeae have been isolated which are highly resistant to tetracycline (MICs of 16 to 64 micrograms/ml). This resistance was due to the acquisition of the resistance determinant tetM, a transposon-borne determinant initially found in the genus Streptococcus and more recently in Mycoplasma hominis, Ureaplasma urealyticum, and Gardnerella vaginalis. In N. gonorrhoeae, the tetM determinant was located on a 25.2-megadalton plasmid. This plasmid arose from the insertion of tetM into the 24.5-megadalton gonococcal conjugative plasmid. The tetM determinant could be transferred to suitable recipient strains of N. gonorrhoeae by both genetic transformation and conjugation. Images

Resistance Mutations in Protease and Reverse Transcriptase Genes of Human Immunodeficiency Virus Type 1 Isolates from Patients with Combination Antiretroviral Therapy Failure

High-density oligonucleotide arrays were used to determine the sequence of the protease (PR) and reverse transcriptase (RT) genes of human immunodeficiency virus type 1 isolates from 35 patients in whom combination therapy that included a protease inhibitor had failed. Isolates had a median of three PR mutations (range, none to six). Three isolates had no known resistance mutations in PR. Twelve isolates (34%) had two or fewer resistance mutations in PR. The most commonly observed PR mutations were L10I, V82A/T/F, and L90M. No mutations were observed at codons 30 or 48. Mutations at RT codons 215 and 184 were observed in the majority of isolates. These data suggest that therapy can fail in some patients with relatively few PR resistance mutations. Clinical failure in the absence of resistance mutations implies inadequate drug exposure due to pharmacologic factors or suboptimal patient adherence to drug therapy.

High-level tetracycline resistance resulting from TetM in strains of Neisseria spp., Kingella denitrificans, and Eikenella corrodens.

Similar to Neisseria gonorrhoeae, tetracycline-resistant isolates of N. meningitidis, Kingella denitrificans, and Eikenella corrodens contained 25.2-megadalton plasmids carrying the TetM determinant. In contrast, tetracycline-resistant N. subflava biovar perflava-N. sicca and N. mucosa isolates carried the TetM determinant in the chromosome.

Mutations causing in vitro resistance to azithromycin in Neisseria gonorrhoeae

In 1999, a cluster of gonococcal isolates exhibiting high Minimal Inhibitory Concentrations (MICs), to azithromycin (2.0-4.0 mg/l) were identified in Kansas City, MO. Isolates were characterized by auxotype/serovar class, lipoprotein (Lip) subtyping and sequencing of the mtrR gene, which has been implicated in decreased azithromycin susceptibility in the gonococcus. Isolates were Pro/IB-3 and contained the 17c Lip subtype. Molecular characterization of the mtrR gene revealed a 153 base pair insertion sequence located between the mtrR/mtrC promoter and the mtrC gene. Some isolates also contained a frame shift within the mtrR gene. Transformation of these mutations into an azithromycin-sensitive recipient strain resulted in transformants with MICs as high as 2.0 mg/l and inactivation of the mtrD gene reduced azithromycin MICs 270-fold. These results demonstrated that the mtr mutations were responsible for the increased MICs in these isolates.

Detection of Neisseria gonorrhoeae Infection by Ligase Chain Reaction Testing of Urine Among Adolescent Women With and Without Chlamydia trachomatis Infection

Background and Objectives: Culture, the conventional method for detection of Neisseria gonorrhoeae, requires invasive sampling and stringent specimen transport conditions. The recently developed ligase chain reaction test (LCR; Abbott Laboratories; North Chicago, IL) allows noninvasive sampling and stable transport conditions, but has not been evaluated with specimens from adolescent populations. Goal of this Study: To perform a comparative evaluation of a commercial LCR test and culture for the diagnosis of N. gonorrhoeae in adolescent women. Study design: Urine and endocervical swab specimens from 330 teenage women seen in two public health adolescent clinics were tested by LCR and culture. For resolution of discordant results, a polymerase chain reaction (PCR) test was developed that directly amplifies N. gonorrhoeae DNA from urine samples processed for LCR. Results: Thirty‐one of 330 (9.4%) cervical specimens were culture‐positive for N. gonorrhoeae, and 30 of 330 (9.1%) urine specimens were positive by LCR. After resolution of 13 discordant results, the sensitivity, specificity, and positive and negative predictive values of LCR for urine were 88.2%, 100%, 100%, 98.7%, respectively, and for culture of endocervical specimens were 82.3%, 98.9%, 90.3% and 98%, respectively. Conclusions: Although more expensive than culture, LCR offers a sensitive means for the detection of N. gonorrhoeae in urine samples and may be useful for this purpose in settings where pelvic examinations are difficult to perform and simultaneous detection of N. gonorrhoeae and Chlamydia trachomatis is advantageous.

Plasmids of serogroup 1 strains ofLegionella pneumophila

Twelve strains ofLegionella pneumophila were tested for the presence of plasmid DNA. Three strains, belonging to serogroup 1, had large plasmids of 83.8×106 daltons, as determined by electron microscopy. A fourth strain, also from serogroup 1, had a similar large plasmid in addition to a smaller plasmid. Restriction analysis of plasmid DNA isolated from the strains with a single size plasmid indicated that the plasmids were structurally very similar. The biologic functions of these plasmids are yet to be determined.

Use of whole-genome sequencing data to analyze 23S rRNA-mediated azithromycin resistance

The whole-genome sequences of 24 isolates of Neisseria gonorrhoeae with elevated minimum inhibitory concentrations (MICs) to azithromycin (≥2.0 µg/mL) were analyzed against a modified sequence derived from the whole-genome sequence of N. gonorrhoeae FA1090 to determine, by signal ratio, the number of mutant copies of the 23S rRNA gene and the copy number effect on 50S ribosome-mediated azithromycin resistance. Isolates that were predicted to contain four mutated copies were accurately identified compared with the results of direct sequencing. Fewer than four mutated copies gave less accurate results but were consistent with elevated MICs.

P4-S1.05 Polymerase chain reaction-based typing of penA genes exhibiting elevated MIC values to cephalosporins in Isolates of Neisseria gonorrhoeae

Objective Develop a PCR based assays for rapid analysis of isolates that exhibit elevated MIC values to penicillin and cephalosporins (MICs of 0.015–0.06 ug/ml to ceftriaxone). These primers were designed to detect mosaic and nonmosaic sequences in the penA gene. Methods Two sets of primer pairs were generated that allowed the detection of mosaic-type and nonmosaic-type penA gene sequences in isolates of N gonorrhoeae that exhibited elevated MIC values to three cephalosporins. Additional primer sets were generated that detected the presence of an inserted aspartic acid residue in nonmosaic penA genes involved in resistance and to detect base substitutions to distinguish between two different mosaic penA genes. DNA sequences were determined as necessary by standard sequencing or pyrosequencing. Results PCR analysis of 28 isolates, exhibiting elevated MIC values to cephalosporins and from different geographic areas, that used these primer sets in combination allowed for the detection of mosaic penA genes in 12 isolates. The remaining isolates possessed nonmosaic genes. Not all of the 12 isolates contained complete mosaic-type sequences with five of the isolates indicating mosaic sequences present in the 5′ region of the penA gene with wild type DNA sequence replacing the last 120 bp of the gene. Another isolate contained nonmosaic penA sequences with an aspartic acid insertion (after aa 345) in the 5′ region of the gene but mosaic sequences in the 3′ end. Finally, mosaic-related sequences were detected by PCR and confirmed by DNA sequencing in the last 106 base pairs of the penA gene of a penicillin sensitive strain isolated prior to 1976. Conclusion PCR can be used to easily detect the presence of mosaic penA genes in isolates of N gonorrhoeae. This study has shown the 3′ region of the gene may contain mosaic, partial mosaic and nonmosaic-type sequences and that mosaic-type sequence has been encountered in the 3′ region of penA in a sensitive strain. We have also observed isolates with increased MICs to cephalosporins that do not contain mosaic sequences. Finally, isolates that possess mosaic penA genes have been detected in dispersed geographic regions in the US, which is of concern due to the evidence mosaic-type penA alleles are involved in the process of increasing gonococcal MICs to cephalosporins.