Posttherapy Isolates Research Articles

Development of resistance in Pseudomonas aeruginosa to imipenem, norfloxacin, and ciprofloxacin during therapy: proof provided by typing with a DNA probe.

Differentiating reinfection from the acquisition of resistance in strains of Pseudomonas aeruginosa after antimicrobial therapy is difficult because currently used epidemiological markers are not stable genetic markers. We previously established that a 741-base pair PstI-NruI restriction fragment upstream from the Exotoxin A structural gene is a sensitive, specific, and stable epidemiological marker for P. aeruginosa. Therefore, we used this fragment as a probe in Southern hybridization to compare pre- and post-therapy isolates of P. aeruginosa. The susceptible and resistant pairs were recovered from multiple sources (including sputum, blood, and urine) from patients treated with various doses of imipenem (n = 15), norfloxacin (n = 6), and ciprofloxacin (n = 4). Southern blot analysis showed identity between the pre- and post-therapy isolates in 23 of the 25 pairs. In the majority of pairs studied, failure to eradicate P. aeruginosa after therapy with imipenem, norfloxacin, and ciprofloxacin was due to the development of resistance rather than to reinfection.

Selection of resistance by piperacillin during Pseudomonas aeruginosa endocarditis.

The mechanisms responsible for emergence of resistance during antimicrobial therapy were investigated in isolates obtained from a patient suffering from Pseudomonas aeruginosa endocarditis. The strain was first isolated from blood cultures obtained upon admission of the patient. It was sensitive to piperacillin, ticarcillin and tobramycin. Piperacillin-tobramycin therapy was therefore instituted and led to a rapid improvement of the patient's condition. Unfortunately, the patient subsequently became febrile again and blood cultures continued to yield P. aeruginosa. However, the organism isolated was now resistant to piperacillin and other penicillins tested. Cell-free extracts of the pre- and post-therapy isolates were analyzed for their beta-lactamase activity. The susceptible pre-therapy strain did not produce significant levels of beta-lactamase. In contrast, the post-therapy strain produced high levels of the enzyme. Induction experiments indicated that the production of beta-lactamase was constitutive in the post-therapy isolate. Thus, piperacillin therapy has led to the selection of resistant mutants which produced high levels of beta-lactamase constitutively. This was associated with relapse of the infection and therapeutic failure.

Characterization of acyclovir-resistant and -sensitive herpes simplex viruses isolated from a patient with an acquired immune deficiency.

Several different genital and non-genital HSV isolates were obtained from a patient with an acquired immune deficiency of unknown aetiology. The patient was initially treated with topical acyclovir (ACV) and later with topical and intravenous ACV. In spite of treatment with antiviral drugs the patient continued to shed virus and to have extensive genital ulcerations. Restriction endonuclease (RE) analyses of the viral DNA revealed that all the isolates had characteristic HSV-2 patterns and that there were three genetically distinct virus groups among the ten isolates tested. Three post-therapy isolates, with the same RE pattern, were found to be devoid of thymidine kinase activity (TKD), highly resistant to ACV in cell culture, but sensitive to vidarabine (ara-A), phosphonoacetate and phosphonoformate. Two of these TKD isolates were obtained during and after topical ACV therapy and before intravenous treatment. Mice inoculated intracerebrally with a lethal dose of each of the three TKD viruses were refractory to ACV, but responded to vidarabine or a combination of ACV and ara-A. Mice inoculated with the TK+ viruses (including the pre-therapy isolate) responded to ACV and/or ara-A treatment. The results indicate that: (i) TKD variants may be produced in humans after topical ACV therapy; (ii) different ACV-resistant or sensitive HSV-2 variants can establish latency at different body sites and reactivate; and (iii) when drug-resistant viruses are isolated from patients with multiple reactivations, the drug in question should not be discontinued, since the patients may also be shedding drug-sensitive virus at a different body site.

5-fluorocytosine resistance in Cryptococcus neoformans.

Isolates of Cryptococcus neoformans from six patients were obtained before and after unsuccessful therapy with 5-fluorocytosine (5-FC). Post-therapy isolates exhibited massive and stable 5-FC resistance. The frequency of drug-resistant mutants in susceptible isolates of C. neoformans was <0.001% (70.4 +/- 17.9 per 10(7) cryptococci), whereas mutant frequencies in resistant isolates approached 100%. Non-drug-induced, spontaneously appearing 5-FC resistant mutants were documented in four susceptible isolates of C. neoformans by use of the statistical method of fluctuation analysis. Mutation rates on these same four isolates ranged from 1.2 x 10(-7) to 4.8 x 10(-7). Total intracellular uptake and incorporation of cytosine-5-(3)H (CyH(3)) and 5-fluorocytosine-2-(14)C (5-FC(14)) into a trichloroacetic acid-insoluble fraction were markedly reduced in six isolates with in vivo-acquired resistance when compared with susceptible pretreatment strains from the same patients. Five of these six isolates also had acquired massive resistance to 5-fluorouracil (5-FU), suggesting that a mutation in the uridine-5'-monophosphate pyrophosphorylase was responsible for drug resistance. The sixth isolate, which remained susceptible to 5-FU, appeared to have a defect in a cytosine-specific permease accounting for 5-FC resistance. A single isolate with in vitro-acquired 5-FC and 5-FU resistance had no reduction in uptake or incorporation of CyH(3) or 5-FC(14). The mechanism of resistance in this isolate is discussed.