Abstract
In vitro susceptibilities to 45 antibiotics were determined for 30 genetically and geographically diverse strains of Yersinia pestis by the broth microdilution method at two temperatures, 28°C and 35°C, following Clinical and Laboratory Standards Institute (CLSI) methods. The Y. pestis strains demonstrated susceptibility to aminoglycosides, quinolones, tetracyclines, β-lactams, cephalosporins, and carbapenems. Only a 1-well shift was observed for the majority of antibiotics between the two temperatures. Establishing and comparing antibiotic susceptibilities of a diverse but specific set of Y. pestis strains by standardized methods and establishing population ranges and MIC50 and MIC90 values provide reference information for assessing new antibiotic agents and also provide a baseline for use in monitoring any future emergence of resistance.
INTRODUCTION
Yersinia pestis is the causative agent of plague, a rare infection in humans that usually appears in the bubonic form due to flea bites. Pneumonic plague is an infection of the respiratory tract which can be contagious and rapidly fatal. This form of plague has an incubation period of 3 to 5 days and a mortality rate near 100% (1, 2). Antibiotic intervention can offer relief but only if started very early in the infection (1). Because of the scattered information on MICs under a variety of nonstandardized testing conditions (3–6) and the lack of comparative data on type strains, we report specific antibiotic susceptibility results from use of the Clinical and Laboratory Standards Institute (CLSI) microdilution broth methodology for 30 strains of Y. pestis. This information will be highly useful as baseline data in the event of wartime or terrorist release and for natural and laboratory-acquired infections.
MATERIALS AND METHODS
The Y. pestis strains used in this study (shown in Table 1) were obtained from the USAMRIID collection and selected to represent established biovars, genotypes, and isotypes (7, 8). Most of the antibiotics were obtained from the U.S. Pharmacopoeia (Rockville, MD) except for ceftriaxone and fusidic acid (Sigma Chemical Co., St. Louis, Mo), cethromycin (Advanced Life Sciences), telithromycin (Sanofi-Aventis), garenoxacin (Schering-Plough), gemifloxacin (Oscient), ertapenem (Merck), faropenem (Replidyne), and tigecycline (Wyeth). Most of the stock solutions (5 mg/ml) were prepared for each drug in the appropriate solvents, based on the current CLSI recommendations (9), and stored until use at −70°C. The concentration of our amoxicillin-clavulanate (2:1) stock was 5 mg/2.5 mg per ml, and that of the co-trimoxazole stock was 5 mg/ml sulfamethoxazole and 0.26 mg/ml trimethoprim (19:1). The MICs were determined by the microdilution method in 96-well plates as previously described (10). Antibiotics were serially diluted 2-fold in 50 μl of cation-adjusted Mueller-Hinton broth (CAMHB). The antibiotic range in the plates was 64 to 0.008 μg/ml based on a final well volume of 100 μl after inoculation. The inocula were prepared by picking several colonies from sheep blood agar (SBA) plates grown for 36 to 48 h at 28°C, suspended, and diluted with CAMHB to a bacterial cell density of 106 CFU/ml (conversion factor, 5 × 108 CFU/ml/unit of optical density at 600 nm [OD600]). To each well of the 96-well plate, 50 μl of this dilution was added for a final inoculum density of approximately 5 × 104 CFU/well (5 × 105 CFU/ml). The plates were incubated at either 28°C or 35°C and visually read at 24 and 48 h. Each MIC was determined in triplicate. Quality control of the antibiotic stocks was verified according to the CLSI methods by using Staphylococcus aureus strain ATCC 29213, Pseudomonas aeruginosa strain ATCC 27853, and Escherichia coli strain ATCC 25922 (9). All bacterial work was carried out under biosafety level 3 (BSL3) laboratory conditions.
TABLE 1.
Y. pestis strain information
| Y. pestis strain | Biovar/genotype/isotypea | Geographic origin |
|---|---|---|
| Colorado 92 | Ort/A/O1 | USA |
| C12 | Ort/A/O1 | USA |
| Antiqua | Ort/A/A3 | Congo |
| Pestoides B | Med/D/P1 | FSUb |
| Pestoides F | Ant/K/P2 | FSU |
| Yeo154 | Ant/F/A1a | Japan |
| Angola | Ant/J/A4 | Angola |
| Java9 | Ort/A/O1 | Indonesia |
| M111(74) | Ort/B/O1 | Madagascar |
| LaPaz | Ort/A/O1 | Bolivia |
| 195P | Ort/A/O1 | India |
| T26 | Ant/–/– | Tanzania |
| KIM 10 | Med/I/M1a | Kurdistan |
| Pestoides E | Ant/M/P2 | FSU |
| RFPBM 19 | Ort/A/O2a | Burma |
| PEXU 429 | Ort/A/O1 | Brazil |
| Yokohama | Ant/F/A1a | Japan |
| Nicholisk 41 | Med/G/M2 | Manchuria |
| Nairobi | Ant/F/A2 | Kenya |
| South Park | Ort/A/O1 | USA |
| Cambodia | Ort/A/– | Cambodia |
| 27 | Ort/–/O2b | Vietnam |
| 31 | Ort/–/O2a | Vietnam |
| 390 | Ort/–/O1 | Israel |
| 590 | Ort/B/O1 | Brazil |
| 25 | Ort/–/O2c | Vietnam |
| 316 | –/A/O2a | Unknown |
| 366 | Med/H/M1b | Yemen |
| Harbin 35 | Med/E/M2 | Manchuria |
| Pestoides C | Med/E/P1 | FSU |
RESULTS
Broth dilution MIC data are presented in Table 2. Because of the instability of the virulence plasmids at higher incubation temperatures (11, 12), determinations at 28°C were also performed. The majority of the MICs were within one well at the two temperatures, with only 17.77% (8/45) of the MIC90s having a two-well difference. Agreements between the two temperatures ranged from 25% to 100%. MIC90 agreements with breakpoints varied more widely, especially compared to the Enterobacteriaceae breakpoints. In general, the Y. pestis strains grew better at 28°C than at 35°C. This was expected since the low calcium response associated with the Yersinia virulence plasmid inhibits growth at the higher temperature.
TABLE 2.
Antibiotic susceptibility of 30 Y. pestis strains
| Antibiotic | Broth dilution MIC data at: |
% agreement | Breakpoint % agreement (35°/28°) | |||||
|---|---|---|---|---|---|---|---|---|
| 35°C |
28°C |
|||||||
| Range | MIC50 | MIC90 | Range | MIC50 | MIC90 | |||
| Amikacin | 0.25–4 | 1 | 2 | 0.25–8 | 2 | 8 | 25 | 12.5/50a |
| Gentamicin | 0.06–1 | 0.5 | 1 | 0.06–2 | 1 | 2 | 50 | 25/50b |
| Netilmicin | 0.12–2 | 0.5 | 1 | 0.06–4 | 0.5 | 1 | 100 | 12.5/12.5a |
| Streptomycin | 1–8 | 2 | 4 | 1–16 | 4 | 8 | 50 | 100/50b |
| Tobramycin | 0.06–1 | 0.5 | 1 | 0.06–2 | 0.5 | 1 | 100 | 25/25a |
| Azithromycin | 1–16 | 4 | 8 | 1–64 | 16 | 32 | 25 | |
| Cethromycin | 0.25–4 | 1 | 2 | 1–16 | 4 | 8 | 25 | |
| Telithromycin | 0.5–8 | 2 | 4 | 0.5–32 | 4 | 16 | 25 | |
| Clarithromycin | 16–>64 | 64 | 64 | 4–>64 | 64 | >64 | 100 | |
| Garenoxacin | 0.015–0.06 | 0.06 | 0.06 | 0.004–0.6 | 0.015 | 0.03 | 50 | |
| Ciprofloxacin | 0.008–0.12 | 0.015 | 0.03 | 0.004–0.03 | 0.008 | 0.015 | 50 | 30/1.5b |
| Gatifloxacin | 0.015–0.12 | 0.06 | 0.06 | 0.004–0.06 | 0.03 | 0.03 | 50 | 30/1.5a |
| Gemifloxacin | 0.004–0.03 | 0.015 | 0.03 | 0.002–0.03 | 0.008 | 0.03 | 100 | 12/12a |
| Levofloxacin | 0.008–0.12 | 0.03 | 0.06 | 0.008–0.06 | 0.03 | 0.03 | 50 | 6/3b |
| Moxifloxacin | 0.015–0.06 | 0.03 | 0.06 | 0.008–0.06 | 0.03 | 0.03 | 50 | |
| Nalidixic acid | 0.25–4 | 2 | 4 | 0.5–2 | 1 | 2 | 50 | 25/12.5a |
| Ofloxacin | 0.015–0.25 | 0.06 | 0.12 | 0.008–0.12 | 0.03 | 0.06 | 50 | 6/3a |
| Sparfloxacin | 0.004–0.06 | 0.015 | 0.03 | 0.002–0.03 | 0.008 | 0.015 | 50 | |
| Novobiocin | 8–>64 | 64 | >64 | 8–>64 | 64 | >64 | 100 | |
| Amoxicillin-clavulanate (2:1) | 0.03–1 | 0.25 | 0.5 | 0.06–0.5 | 0.25 | 0.25 | 50 | 6/6a |
| Amoxicillin | 0.03–1 | 0.25 | 0.5 | 0.06–1 | 0.25 | 0.5 | 100 | |
| Ampicillin | 0.03–4 | 0.25 | 0.5 | 0.06–0.5 | 0.25 | 0.5 | 100 | 6/6a |
| Penicillin G | 0.012–1 | 0.25 | 1 | 0.03–2 | 0.5 | 2 | 50 | |
| Piperacillin | 0.03–0.5 | 0.06 | 0.25 | 0.03–0.5 | 0.12 | 0.5 | 50 | 2/3a |
| Imipenem | 0.12–1 | 0.25 | 0.5 | 0.06–1 | 0.25 | 0.5 | 100 | |
| Ertapenem (Invanz) | 0.004–0.06 | 0.03 | 0.03 | 0.002–0.03 | 0.015 | 0.03 | 100 | |
| Faropenem | 0.06–2 | 0.25 | 0.5 | 0.25–1 | 0.5 | 1 | 50 | |
| Meropenem | 0.015–0.12 | 0.06 | 0.12 | 0.015–0.06 | 0.06 | 0.06 | 50 | |
| Cefepime | 0.008–0.06 | 0.03 | 0.06 | 0.03–0.25 | 0.03 | 0.25 | 25 | 1/3a |
| Ceftazidime | 0.03–0.5 | 0.12 | 0.25 | 0.03–0.25 | 0.06 | 0.12 | 50 | 3/1.5a |
| Cefotaxime | 0.004–0.03 | 0.015 | 0.03 | 0.004–0.015 | 0.008 | 0.015 | 50 | 0.4/19a |
| Cefotetan | 0.03–0.5 | 0.25 | 0.5 | 0.03–0.25 | 0.12 | 0.25 | 50 | 3/1.5a |
| Cefuroxime | 0.03–2 | 0.5 | 2 | 0.06–2 | 0.5 | 1 | 50 | 25/12.5a |
| Cefazolin | 0.25–8 | 2 | 4 | 1–4 | 2 | 4 | 50 | 8/25a |
| Ceftriaxone | 0.008–0.03 | 0.015 | 0.03 | 0.008–0.03 | 0.015 | 0.015 | 50 | 0.4/19a |
| Aztreonam | 0.008–0.06 | 0.03 | 0.03 | 0.008–0.03 | 0.015 | 0.03 | 100 | 0.4/38a |
| Sulfamethoxazole | 0.5–>64 | 16 | 64 | 2−>64 | 16 | >64 | 100 | 25/25a |
| Sulfamethoxazole-trimethoprim (19:1) | 0.25–8 | 0.5 | 8 | 0.12–4 | 1 | 4 | 50 | 21/11b |
| Trimethoprim | 0.12–16 | 0.5 | 8 | 0.12–8 | 0.5 | 2 | 25 | 1/25a |
| Doxycycline | 0.06–2 | 0.5 | 1 | 0.06–0.5 | 0.25 | 0.5 | 50 | 25/12.5b |
| Tetracycline | 0.25–2 | 0.5 | 2 | 1–16 | 4 | 8 | 25 | 50/50b |
| Tigecycline | 0.06–0.5 | 0.25 | 0.25 | 0.03–0.5 | 0.12 | 0.5 | 50 | |
| Rifampin | 0.25–4 | 2 | 4 | 1–64 | 2 | 16 | 25 | |
| Chloramphenicol | 0.25–4 | 1 | 4 | 0.5–16 | 4 | 8 | 50 | 50/100b |
| Fusidic acid | 16–>64 | 64 | >64 | 2–>64 | 32 | 64 | 100 | |
DISCUSSION
The establishment of MICs for a number of defined and archived strains of Y. pestis will be helpful in serving as references in future testing. Few susceptibility breakpoints have been established for Y. pestis. The CLSI has developed some interpretive criteria based in part on these data, other published in vitro distribution data, and animal efficacy data (13). Standard testing at 35°C provided MIC90s that indicate susceptibility to gentamicin, streptomycin, doxycycline, tetracycline, ciprofloxacin, levofloxacin, co-trimoxazole, and chloramphenicol based on those breakpoints (13), and these values agree with reference development data and data from a recent 392-strain study (10, 14). If the available Enterobacteriaceae breakpoints are used (15), most of the MIC90s for the antibiotics evaluated would be considered susceptible. There are no breakpoints for the macrolides, novobiocin, or fusidic acid. However, the MIC90 values suggest that none of these antibiotics would be in a susceptible range, with the possible exception of cethromycin (MIC90, 2 μg/ml). In a rat model, cethromycin showed some efficacy but only at very high doses that were well above the proposed human dose of 300 mg/day (16). Similar results have been observed in the mouse pneumonic plague model (H. S. Heine, unpublished data). These results suggest that an MIC of 1 to 2 μg/ml is higher than the efficacious breakpoint for Y. pestis. While the β-lactam and cephalosporin antibiotics demonstrate very good in vitro activity, animal efficacy data indicate that use of these antibiotics should be contraindicated (17). The equivalent results observed at 28°C may be significant. It was previously shown that the virulence plasmids in Y. pestis are unstable when grown at temperatures ranging from 35°C to 37°C (11, 12), which is the CLSI standard incubation temperature. While antibiotic resistance has not yet been associated with the virulence plasmids, the recent isolation of several Y. pestis strains containing multiple antibiotic resistance genes with transposable sequences (18, 19) raises the possibility of gene transfer to the virulence plasmids. To adequately assess the susceptibility profile of such a recombinant strain, to maintain the plasmid stability, and to obtain a reliable antibiotic reading, incubation at 28°C might be warranted. The observation in this study that susceptibility values did not shift significantly should be useful for reference laboratories.
ACKNOWLEDGMENTS
Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army or the University of Florida.
The research described herein was sponsored by the Defense Threat Reduction Agency project no. 02-4-2C-013.
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