We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147-151, 1974), and the entire pncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were >100 g/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a threecodon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 g/ml, 3 had the same mutation (Thr473Ala) and 18 had the wild-type sequence. For the three Thr473Ala mutants PZA MICs were 12.5 g/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 g of PZA per ml and the third was resistant to 800 g of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to >100 g of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr473Ala mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to >100 g of PZA per ml.

Antimicrobial susceptibility patterns of 102 clinical isolates of Serratia marcescens from three medical centers were studied by using disk sensitivity and agar dilution methods. The least resistance was demonstrated against gentamicin, nalidixic acid, chloramphenicol, and sulfisoxazole, all of which inhibited more than 80% of the strains. Cephalothin was completely ineffective, and more than 90% of strains were resistant to ampicillin and tetracycline. As demonstrated by the agar dilution method, the minimal inhibitory concentration of nalidixic acid, gentamicin, tobramycin, and chloramphenicol for most strains fell within therapeutically attainable concentrations. The prevalence of resistance to ampicillin, cephalothin, and tetracycline was nearly the same at all three medical centers, whereas there appeared to be patterns characteristic for each center with regard to the other drugs used. Eleven of the isolates produced pigment and exhibited patterns similar but not identical to those of the nonpigmented strains, all 11 being resistant to between three and six drugs. Half of the strains were resistant to five or more antibiotics, indicating that some Serratia exhibit resistance to an unusually broad range of therapeutic agents.

The infectious disease applications of nucleic acid probe have been described. In addition, the basic procedures of nucleic acid probe technology have been discussed, as have the factors affecting implementation of probe technology in diagnostic laboratories. Despite the questions raised, nucleic acid probes will become part of the diagnostic laboratory in the near future. Commercial interests are developing and marketing new probes, reagents, and kits which will expedite the employment of this technology. High-volume reference laboratories will first use probes as part of a battery of tests which will include ELISA and monoclonal antibody methods. In all probability, probes will replace methods: that have proven to be ineffective, difficult, or costly such as culturing for some enteric pathogens and Legionella, that require long incubation periods, such as mycobacteria, or that have high costs and low yields, such as virology.