Identification of the Conjugative mefGene in Clinical Acinetobacter junii and Neisseria gonorrhoeae Isolates

Bacteria.Erythromycin-resistant (Ery^r)Acinetobacter junii strain 329 was isolated from the oral gumline of a child in Portugal (Table 1). The organism was identified by D. Stroman (Alcon Laboratories, Inc., Fort Worth, Tex.) by sequencing the 16S rRNA. Sixteen N. gonorrhoeae isolates with variable susceptibilities to erythromycin (MIC = 0.25 to 4 μg/ml) were collected from urethral specimens from adults in Seattle, Wash., and identified by theNeisseria Reference Laboratory at the University of Washington (Table 1). Thirteen N. gonorrhoeae isolates carrying the tetM 25.2-MDa plasmid were collected in Seattle and the eastern United States during 1983 through 1986 and have been previously described (7). Of the three Ery^r S. pneumoniae strains carrying the mef gene, two (n011 and 970146) were isolated in Washington State and the third (02J1048) was supplied by Pfizer Inc. (Groton, Conn.) (Table 1) (9, 26). Strains used as recipients are listed in Table2. Antibiotic susceptibilities were determined using either disk diffusion or agar dilution as described by the NCCLS (13).

Media. S. pneumoniae isolates were grown on brucella blood agar (Difco, Detroit, Mich.) supplemented with 5% sheep red blood cells and incubated with 5% CO₂ at 36.5°C (8). The Haemophilus influenzae isolate was grown on brain heart infusion (BHI) agar (Difco) supplemented with 2 μg of NAD (Sigma Chemical Co., St. Louis, Mo.) per ml and 10 μg of hemin–l-histidine (Sigma Chemical Co.) per ml (16). Eikenella corrodens, Kingella denitrificans, Moraxella catarrhalis, N. gonorrhoeae, Neisseria meningitidis, Neisseria perflava/sicca, and Neisseria mucosa were grown on gonococcus (GC) medium agar (Difco) supplemented with Kellogg's supplement solution (0.22 M d-glucose, 0.03 Ml-glutamine, 0.001 M ferric nitrate, and 0.02 M cocarboxylase) (Sigma-Aldrich Chemical Co., St. Louis, Mo.) as previously described (6, 15). Enterococcus faecalis JH2-2 was grown on BHI agar without supplements (9). For DNA dot blots and DNA extractions, the gram-positive bacteria were grown overnight at 36.5°C in BHI broth (Difco) supplemented with 0.03 M d-glucose and 0.04%dl-threonine (8, 9). The gram-negative bacteria, for DNA dot blots and DNA extractions, were grown in either BHI broth with no supplements (A. junii), BHI broth supplemented with 2 μg of NAD per ml and 10 μg of hemin–l-histidine per ml (H. influenzae), or GC broth supplemented with 1% sodium bicarbonate and Kellogg's supplement (see above) (E. corrodens, K. denitrificans, M. catarrhalis, and Neisseria spp.) as described previously (11, 12,15).

Mating procedure.Donor and recipient bacteria were grown separately at 36.5°C overnight. Each isolate was suspended in 0.5 to 1.0 ml of BHI or GC broth (Difco) to a density of approximately 10⁹ cells per ml (3 McFarland). Donor and recipient bacteria at ratios of 1:20 or 1:40 (donor to recipient) were mixed. The bacterial suspension was then plated directly onto brucella blood agar plates and incubated in CO₂ at 36.5°C for 48 h (8, 9). After incubation, the mating mixture was serially diluted onto antibiotic-supplemented plates as follows: for H. influenzae recipients, erythromycin (10 μg/ml) and rifampin (10 μg/ml); for E. faecalis recipients, erythromycin (10 μg/ml) and rifampin (10 μg/ml); for M. catarrhalis,N. gonorrhoeae, and N. meningitidis recipients, erythromycin (2 μg/ml) and rifampin (10 μg/ml); and for E. corrodens, K. denitrificans, N. perflava/sicca, and N. mucosa recipients, erythromycin (2 μg/ml) and tetracycline (10 μg/ml). Gram stains were prepared from all transconjugants, and all transconjugants were identified to species level by established methods (4, 14). Mating experiments were performed a minimum of two times. DNase I (1 mg/ml) (Sigma-Aldrich Chemical Co.) was added to the mating mixture in at least one of the two matings for comparison with mating without DNase to rule out transformation.

Extraction of whole-cell DNA.Whole-cell DNA was prepared from isolates and transconjugants as previously described (1,8). DNA was separated on a 0.7% agarose gel and Southern blots were prepared (22).

Plasmid analysis of transconjugants.Plasmid extractions were prepared from K. denitrificans, N. perflava/sicca, and their respective transconjugants after 8 h of growth in 40 ml of GC peptone broth supplemented with Kellogg's supplement solution and 1% sodium bicarbonate as previously described (3, 15). DNA was separated on a 0.7% agarose gel in 0.5× Tris-borate-EDTA (TBE) buffer at 100 V for 1.5 h; Southern blots were prepared and hybridized with mef-specific³²P-labeled oligonucleotide probe MF5 to examine the location of the mef gene.

Dot blots.Two milliliters of overnight bacterial growth in supplemented BHI or GC broth was placed into 1.5-ml sterile Eppendorf tubes and centrifuged at 8,000 × g for 2 min, and the supernatant was decanted. The bacterial pellet was resuspended with 1 ml of BHI broth to create a turbid suspension of 3 McFarland. Two hundred microliters of the suspension, in 25-μl aliquots, was spotted onto a GeneScreen Plus membrane (NEN Research, Boston, Mass.), dried, and treated with 0.5 M NaOH for 10 min, 1 M Tris-HCl for 3 min, and 1 M Tris-HCl with 1.5% NaCl, pH 7.5, for 10 min. The membrane was dried, washed in chloroform-isoamyl alcohol (24:1), rinsed in water twice, washed in 1 M Tris-HCl with 1.5% NaCl for 10 min, and baked at 80°C for 1 h. The filters were stored at room temperature until hybridized with labeled oligonucleotide probes (8).

Labeled probes.MF5 (5′ GGT GCT GTG ATT GCA TCT ATT AC 3′) was used as the oligonucleotide probe for the mef gene (9). The oligonucleotide probes for the erm genes were the following: ermB_F (5′ GAA AAG GTA CTC AAC CAA ATA 3′), ermC_R (5′ GCT AAT ATT GTT TAA ATC GTC AAT 3′), and ermF1 (5′ CGG GTC AGC ACT TTA CTA TTG 3′) (19). A³²P-labeled probe was used for whole-bacterial-cell dots as previously described (9).

PCR.PCR amplification used 40 ng of genomic DNA fromS. pneumoniae 02J1048 as a positive control and 30 ng of genomic DNA as a template from the transconjugants and A. junii. Primers were MF4a (5′ ACC GAT TCT ATC AGC AAA G 3′) and MF6 (5′ GGA CCT GCC ATT GGT GTG 3′). Both primers are in the conserved regions of the mef gene. Each reaction mixture contained 2 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, Conn.), 200 μM deoxynucleoside triphosphates, 1× PCR buffer (1.5 mM MgCl₂), and 100 ng of each primer. Using a Perkin-Elmer Cetus thermal cycler, the reactions were carried out by denaturation at 94°C for 1 min, annealing at 37°C for 1 min, and elongation at 72°C for 2 min, for 35 cycles. The PCR products were dried, resuspended in 1/10 volume of sterile water, and separated on a 1.5% agarose gel with 0.5× TBE running buffer. The PCR bands were visualized by ethidium bromide staining, and Southern blots were prepared. The 940-bp PCR product hybridized with a labeled internalmef probe, MF5 (9). Positive and negative controls were run with each assay.

DNA-DNA hybridization.Southern blots using uncut whole-cell DNA or the PCR fragments were prepared on Magnagraph nylon (Micron Separation Inc., Westboro, Mass.) and hybridized with³²P-labeled oligonucleotide MF5. DNA dot blots containing 30 to 300 μg of extracted whole-cell DNA from the donor strains and bacterial dot blots from transconjugants were placed on GeneScreen Plus membranes and hybridized with the radiolabeled oligonucleotide probe (8, 9).

Sequencing.Sequencing of selected isolates (A. junii 329 and N. gonorrhoeae 98.420 and 98.737) was performed using oligonucleotide primers MF7 (5′ ATG CAG ACC AAA AGC GCG AT 3′) and MF8 (5′ CGG TAT CTG TTC TGG TAG CG 3′). Both primers are in the conserved regions of the mef gene and within the generated PCR fragment. Each reaction mixture contained 2 U ofTaq polymerase, 200 μM deoxynucleoside triphosphates, 1× PCR buffer (1.5 mM MgCl₂), and 100 ng of each primer. Using a Perkin-Elmer Cetus thermal cycler, the reactions were carried out by denaturation at 94°C for 1 min, annealing at 51°C for 1 min, and elongation at 72°C for 2 min, for 35 cycles. The PCR products were filtered by centrifugation with a 0.5-μm-pore-size separation filter (Micron) by following the manufacturer's directions. A small part of the PCR product obtained was dried and separated on a 1.5% agarose gel with 0.5× TBE running buffer. The 253-bp PCR product was then visualized by ethidium bromide staining. The remainder of the PCR product was then sequenced by following the Big Dye Terminator chemistry protocol (Perkin-Elmer Cetus, Foster City, Calif.). The sequence reaction mixture included 100 ng of the PCR product and 4 pmol of primer MF7, with a total volume of 12 μl. The mefsequence was compared to the mef sequence from S. pneumoniae (GenBank number U83667) using the Genetics Computer Group software (University of Wisconsin, Madison).