Antibiotic Resistance of Y. pestis 17/95

Table 2. Table 2. Minimal Inhibitory Concentrations of Various Antibiotics against the Bacterial Strains Studied.

Disk-agar diffusion tests showed that Y. pestis 17/95 was resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, spectinomycin, sulfonamides, tetracycline, and minocycline. Resistance to ampicillin was due to the production of a beta-lactamase, and resistance to chloramphenicol was due to the production of a chloramphenicol acetyltransferase. Resistance to kanamycin was due to synthesis of a type I 3'-aminoglycoside phosphotransferase. The strain was also resistant to high levels of streptomycin–spectinomycin as a result of the production of 3߱-9-aminoglycoside adenylyltransferase. Y. pestis 17/95 was resistant to sulfonamides6 but remained susceptible to trimethoprim (Table 2), and no synergism was detected between the two drugs by the checkerboard method.16

A small percentage of Y. pestis 17/95 spontaneously lost the resistance determinants en bloc (1 of 100 colonies tested after 10 days of incubation in the absence of antibiotics), and this clone, 17/95-I, was studied further.

All the resistance genes were transferred by conjugation from Y. pestis 17/95 to avirulent Y. pestis 6/69cN at a frequency of 1.5×10-2 per donor colony-forming unit. Selection for transfer of one of these resistance characters revealed the transfer of all six. The minimal inhibitory concentrations of antibiotics for the parent strain, the clone that had lost the resistance determinants, strain 6/69cN, and a strain obtained by the conjugation of 17/95 with 6/69cN are shown in Table 2.

Figure 1. Figure 1. Analysis of Plasmid DNA by Agarose-Gel Electrophoresis (Panel A) and Hybridization (Panel B). Plasmid DNA from Y. pestis 6/69 (lane 1), 17/95 (lane 2), and 17/95-I (lane 3) and E. coli BM4359 (lane 4) was digested with EcoRV, fractionated by agarose-gel electrophoresis (Panel A), transferred to a nitrocellulose sheet, and hybridized to the 32P-labeled te t(D) PCR product (Panel B). Lane 5 shows fragments obtained by the digestion of bacteriophage lambda DNA with Hi ndIII and used as standards for molecular size.

Plasmid DNA from Y. pestis 6/69, 17/95, and 17/95-I was extracted and digested with EcoRV (Figure 1A, lanes 1, 2, and 3). Comparison of the restriction profiles indicated that strain 17/95 contained fragments corresponding to an additional plasmid, designated pIP1202, of approximately 150,000 bp, as estimated by pulsed-field gel electrophoresis (data not shown).

Characterization of Plasmid pIP1202

Plasmid pIP1202 was transferred by conjugation from Y. pestis 17/95 to E. coli K802N and RR1 at frequencies of approximately 1 ×10-2.The minimal inhibitory concentrations of antibiotics for E. coli K802N and strain BM4354, obtained by conjugation of Y. pestis 17/95 with E. coli K802N, are shown in Table 2. The retransfer of pIP1202 from E. coli BM4359 to Y. pestis 6/69cN and E. coli K802N occurred at frequencies of 1.1 × 10-4 and 5.7 × 10-5, respectively.

Approximately 5 percent of E. coli BM4354 did not contain plasmid pIP1202. Approximately 1 percent had lost part of the resistant determinants, generating plasmids pIP1202-1, -2, -3, and -4 (in Table 1), which were used to assess the incompatibility of the plasmids in experiments performed by reciprocal conjugation. Plasmid pIP1202-2 exhibited strong incompatibility with pIP55-1, which belongs to the Inc6-C group.17 Hybridization with a probe specific for Inc6-C replicons18 was detected only with plasmid DNA from the parental strain 17/95 and the E. coli strain that had acquired pIP1202 by conjugation from 17/95, confirming that this plasmid belongs to incompatibility group Inc6-C (data not shown).

Analysis of Plasmid DNA