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A real-time PCR assay identified linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolates with a G2576U rRNA mutation. PCR-restriction fragment length polymorphism analysis of ribosomal DNA amplicons with NheI also detected this mutation. Both assays detected isolates heterozygous at this position. Recognition of isolates with what is presently the most frequent oxazolidinone resistance mutation may aid surveillance and individual case management.

This study is focused on real-time detection of gyrA mutations and of the presence of class I integrons in a panel of 100 veterinary isolates of Salmonella enterica from farm animals. The isolates were selected on the basis of resistance to nalidixic acid, representing a variety of the most prevalent serotypes in England and Wales. In addition, organic solvent (cyclohexane) resistance in these isolates was investigated in an attempt to elucidate the presence of efflux pump mechanisms. The most prevalent mutation among the isolates studied was Asp87-Asn (n = 42), followed by Ser83-Phe (n = 38), Ser83-Tyr (n = 12), Asp87-Tyr (n = 4), and Asp87-Gly (n = 3). Two distinct subpopulations were identified, separated at the 1-mg/liter breakpoint for ciprofloxacin: 86% of isolates with mutations in codon 83 showed MICs of >or=1 mg/liter, while 89.8% of isolates with mutations in codon 87 presented MICs of or=2.0 mg/liter. Thirty-four isolates contained class I integrons, with 71% of the S. enterica serovar Typhimurium isolates and 6.9% of isolates belonging to other serotypes containing such elements. The methods used represent sensitive ways of investigating the presence of gyrA mutations and of detecting class-I integrons in Salmonella isolates. The results can be obtained in less than 1 h from single colonies without the need for purifying DNA.

A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC-->AAC) mutation, an Asp-87-to-Gly (GAC-->GGC) mutation, and a Ser-83-to-Phe (TCC-->TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC-->TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC-->TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.

A nucleic acid-based test (Gen-Probe PACE 2C System) was evaluated for the ability to detect Chlamydia trachomatis and Neisseria gonorrhoeae from endocervical specimens in a single assay. Three swab samples, randomized for collection order, were obtained from each patient and tested by N. gonorrhoeae and C. trachomatis culture and by the PACE 2C probe assay. Fifty of 395 specimens were culture positive for N. gonorrhoeae (17 specimens), C. trachomatis (26 specimens), or both (7 specimens), of which PACE 2C testing detected 48 specimens. The PACE 2C assay was positive for 56 specimens, including 8 specimens not positive by culture. Of the total of 10 discrepancies between culture and PACE 2C results, resolution testing yielded four false-negative culture, four false-positive PACE 2C, and two false-negative PACE 2C results. The sensitivity, specificity, and positive and negative predictive values for PACE 2C after reevaluation were 96.3, 98.8, 92.9 and 99.4%, respectively. The overall sensitivities for C. trachomatis and N. gonorrhoeae culture were 89.2 and 88.9%, respectively. The prevalence rate for C. trachomatis was 9.4%, and that for N. gonorrhoeae was 6.8%. The Gen-Probe PACE 2C System is a reliable alternative for screening endocervical specimens for both C. trachomatis and N. gonorrhoeae in a single assay.

Highly purified polymeric flagellin preparations from 10 different Salmonella serotypes were used to produce specific Salmonella H antisera with high titers by the immunization of rabbits. Antigen emulsions in complete Freund adjuvant were administered at the rate of 50 micrograms per rabbit by multiple intradermal injection. Booster injections were given 110 days after the primary immunization. The immune response was monitored regularly over a period of 200 days. The results showed that the H titers, determined with 125I-labeled antigens, averaged 61,000 +/- 39,000, and the H agglutination titers of 83% of the animals were greater than 40,000. The high titers of the immunized animals persisted for approximately 4 months. The O agglutination titers of the antisera were less than 10 for 9 of the flagellin preparations and ranged from 10 to 320 for the remaining preparation. The antisera obtained were serotype specific after appropriate dilution.

Ten different Salmonella serotypes were grown in a chemically defined medium supplemented with 0.01% yeast extract. After sedimentation of the cells by centrifugation, flagella were detached by exposure to pH 2 for 30 min at room temperature. The flagellaless cells were removed by centrifugation, and the flagellin in the supernatant was further purified by high-speed centrifugation, ammonium sulfate precipitation, and dialysis in 50,000-molecular-weight-cutoff tubing. The 10 flagellin preparations were of a high degree of purity, as demonstrated by electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of salmonella H and O agglutination titers of antisera raised in rabbits with the flagellin preparations as immunogens.

Several cultures of gram-negative and gram-positive bacteria were successfully immobilized with titanous hydroxide. The immobilization efficiency for the microorganisms investigated in saline and broth media ranged from 80.2 to 99.9%. The immobilization of salmonellae was effective over a wide pH range. The presence of buffers, particularly phosphate buffer, drastically reduced the immobilization rate. However, buffers may be added to immunoassay systems after immobilization of microorganisms. The immobilization process involved only one step, i.e., shaking 100 microliter of culture with 50 microliter of titanous hydroxide suspension in polystyrene tubes for only 10 min. The immobilized cells were so tenaciously bound that vigorous agitation for 24 h did not result in cell dissociation. The nonspecific binding of 125I-labeled antibody from rabbits and 125I-labeled protein A by titanous hydroxide was inhibited in the presence of 2% gelatin and amounted to only 5.6 and 3.9%, respectively. We conclude that this immobilization procedure is a potentially powerful tool which could be utilized in solid-phase immunoassays concerned with the diagnosis of microorganisms.