Antibiotic susceptibilities of 94 isolates of Yersinia pestis to 24 antimicrobial agents

Eric Hernandez1,*, Monique Girardet1, Francoise Ramisse2, Dominique Vidal3 and Jean-Didier Cavallo1

1 Hôpital d’iInstruction des armées Bégin, Biologie médicale, Saint-mandé; 2 Centre d’études du Bouchet, Laboratoire de microbiologie, Vert-le-petit; 3 Centre de recherches du service de santé des armées, Laboratoire de microbiologie, La tronche, France

Received 10 April 2003; returned 21 May 2003; revised 13 August 2003; accepted 25 September 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Ninety-four isolates of Yersinia pestis collected by the French army between 1964 and 1988 were evaluated for their susceptibilities to 24 antibiotics by the agar dilution method. All the isolates were susceptible to ß-lactam antibiotics including imipenem, to fluoroquinolones, aminoglycosides and to doxycycline. The most active compounds were fluoroquinolone antibiotics, third-generation cephalosporins and aminoglycosides.

Keywords: Y. pestis, antibiotic susceptibility, plague


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Plague is a zoonotic disease affecting rodents. Transmission to humans occurs primarily following the bites of fleas from infected animals, and infection is usually manifested as one of the three primary forms: bubonic, septicaemic or pneumonic. Mortality for untreated bubonic plague is higher than 40%, whereas untreated septicaemic or pneumonic forms are usually fatal. Despite the efforts to control the disease, plague remains enzootic in Asia, Central-Asia, Africa, Madagascar and both in North and South America. Because of its high pathogenicity and ability to cause epidemics, Yersinia pestis has been listed among the several agents likely to be used as a biological weapon in a bioterrorism event by the Centers for Disease Control (CDC).

Streptomycin is the drug of choice for the treatment of infection in humans but is no longer available in some countries. Tetracycline and chloramphenicol have been demonstrated to be effective alter-natives in humans.1 In animal models, doxycycline, ciprofloxacin and ofloxacin have demonstrated their effectiveness, but have not been employed as first-line options for plague treatment in humans. Penicillins and cephalosporins are not effective in vivo.2 Resistance to antibiotics is exceptional, but a high level of resistance to multiple antibiotics as a result of a self-transferable plasmid has been observed in a strain isolated from a patient with bubonic plague.3 Another isolate, highly resistant to streptomycin has also been described.4 Furthermore, Wong et al. published a percentage of 20.6% resistance to imipenem among 92 strains isolated over a 21-year period in the United States.5

As described by Inglesby et al.,6 the use of an aerosolized plague weapon could cause fever, cough, chest pain and haemoptysis with signs consistent with severe pneumonia 1–6 days after exposure. Rapid evolution of disease would occur in the 2–4 days after symptom onset and would lead to septic shock with high mortality without early treatment.6 Tetracycline, streptomycin, gentamicin and fluoroquinolone antibiotics have been recommended for post-exposure prophylaxis.6 There is, however, a need to continue to carry out in vitro susceptibility testing to identify potential alternative agents for the treatment of plague.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Security

All the experiments were conducted in the BSL3 laboratory of the Centre d’Etudes du Bouchet (Vert-le-petit, France), which is part of the French Ministry of Defence.

Bacterial strains

Ninety-four isolates of the collection of the Centre d’Etudes du Bouchet (Vert-le-petit, France) were included in this protocol. Bacteria are stored in the BSL3 laboratory of the institute. Isolates were collected between 1964 and 1988 and were from Saigon (12, isolated in 1964), Dalat (45 isolated in 1966, 16 in 1967 and five in 1968), Madagascar (two isolated in 1981, and three in 1988), Kurdistan (one, isolated in 1979), Birmania (two, isolated in 1979), Java (two, isolated in 1979) and India (two, isolated in 1979). The other strains tested were CIP 6/69M, the vaccinal strain EV76 and two strains provided by the Pasteur Institute of Lille, France. Among these strains six were medievalis, six were orientalis type 1, and the other strains were orientalis type 2. Typing was done as described by Motin et al.7 The host of origin is unknown.

Susceptibility testing

Antibiotics tested were: amoxicillin (Inava, France), co-amoxiclav (clavulanic acid 2 mg/L) (Smith-Kline-Beecham, France), piperacillin (Dakota, France), piperacillin/tazobactam (tazobactam 4 mg/L) (Wyeth-Lederle, France), cefalothin (Panpharma, France), cefoxitin (Merck-Sharp-Dohme-Chibret, France), cefotaxime (Roussel-Diamand, France), ceftazidime (Glaxo-Wellcome, France), aztreonam (Sanofi-Winthrop, France), imipenem (Merck-Sharp-Dohme-Chibret, France), nalidixic acid (Sanofi-Winthrop, France), ofloxacin (Roussel-Diamand, France), pefloxacin (Bellon, France), ciprofloxacin (Bayer, France), norfloxacin (Glaxo-Wellcome, France), gatifloxacin (Grunenthal, France), trimethoprim/sulfamethoxazole (Roche, France), streptomycin (Pharmacie centrale des armies, France), gentamicin (Schering-Plough), amikacin (Bristol-Myers-Squibb, France), tobramycin (Lilly, France), doxycycline (Asta-medica, France), chloramphenicol (Chauvin, France) and colistin (Bellon, France). The concentrations of ß-lactamase inhibitors described were used with all dilutions of amoxicillin and piperacillin.

Antimicrobial agents were reconstituted according to manufacturers’ recommendations. Susceptibility testing was done by the agar-dilution method in Mueller–Hinton medium. After identification by routine laboratory technique (Biolog system, Hayward, CA, USA), three or four colonies of each isolate were plated on blood agar and incubated for 48 h at 28°C. Colonies from plates were then suspended in PBS in order to obtain a final inoculum of 108 cfu/mL. One hundred microlitres was added to 3.9 mL of Mueller–Hinton broth and incubated for 24 h in a water bath at 28°C. Final inoculum was prepared before inoculation and was 104 cfu/spot. After inoculation, agar-plates were incubated for 48 h at 28°C. Two operators read minimal inhibitory concentrations (MICs). Quality control was Escherichia coli ATCC 25922. Interpretative criteria were done according to the Comité de l’Antibiogramme de la Société Française de Microbiologie.8 These recommendations are freely available at http://www.sfm.asso.fr.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
All the isolates tested in this protocol were susceptible to ß-lactams, aminoglycosides and fluoroquinolone antibiotics (Table 1). This result correlates well with the data previously published that demonstrated that Y. pestis isolates are usually susceptible to many antibiotics.9 There was no difference in MICs between isolates from various geographical areas. Lower MICs were obtained with fluoroquinolone antibiotics and third-generation cephalosporins (Table 1). Among the fluoroquinolones, the MICs of norfloxacin were higher than those observed with ofloxacin, pefloxacin and ciprofloxacin. This has been described for other Enterobacteriaceae. Gatifloxacin, a new fluoroquinolone, had the same activity as ciprofloxacin. MICs of piperacillin were equivalent to those of amoxicillin. Cefalothin and cefoxitin were effective but MICs of these antibiotics were higher than those observed with the other ß-lactams.


View this table:
[in this window]
[in a new window]
 
Table 1. Susceptibility testing of 94 isolates of Y. pestis
 
All the isolates were susceptible to aztreonam and there was no resistant or intermediate strain to imipenem. This result is different to the result obtained by Wong et al.5 who described 20.6% resistance among 92 strains tested in 1999. All the strains were susceptible to chloramphenicol but this antibiotic has been described as less effective in reducing mortality than standard therapy.10 Only 30 isolates were susceptible to colistin, the others were intermediate or resistant. Among the susceptible strains 14 had MICs between 1 and 2 mg/L (breakpoint for susceptibility <= 2 mg/L). This result, however, is not of significant importance because this antibiotic is not used either in prophylaxis or for the treatment of plague infection and must be confirmed with Y. pestis from other collections.

In conclusion, Y. pestis remains susceptible to most antibiotics tested (except colistin) with a higher efficacy for fluoroquinolones, third-generation cephalosporins and aminoglycosides. All the strains tested were susceptible to the antibiotics recommended for post-exposure prophylaxis. However, further in vivo studies are needed for determining alternative antibiotic treatments in case of bioterrorist attack with strains resistant to recommended antibiotics.


    Acknowledgements
 
This work was supported by the French Ministry of Defence.


    Footnotes
 
* Corresponding author. Tel: +33-1-43-98-47-33; Fax: +33-1-43-98-53-36; E-mail: hnz.eric{at}freesurf.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Butler, T. (1989). The black death past and present. Plague in the 1980s. Transactions of the Royal Society of Tropical Medicine and Hygiene 83, 458–60.[ISI][Medline]

2 . Byrne, W. R., Welkos, S. L., Pitt, M. L. et al. (1998). Antibiotic treatment of experimental pneumonic plague in mice. Antimicrobial Agents and Chemotherapy 42, 675–81.[Abstract/Free Full Text]

3 . Galimand, M., Guiyoule, A., Gerbaud, G. et al. (1997). Multidrug resistance in Yersinia pestis mediated by a transferable plasmid. New England Journal of Medicine 337, 677–80.[Free Full Text]

4 . Guiyoule, A., Gerbaud, G., Buchrieser, C. et al. (2001). Transferable plasmid-mediated resistance to streptomycin in a clinical isolate of Yersinia pestis. Emerging Infectious Diseases 7, 43–8.[ISI][Medline]

5 . Wong, J. D., Barash, J. R., Sandfort, R. F. et al. (2000). Susceptibilities of Yersinia pestis strains to 12 antimicrobial agents. Antimicrobial Agents and Chemotherapy 44, 1995–6.[Abstract/Free Full Text]

6 . Inglesby, T. V., Dennis, D. T., Henderson, D. A. et al. (2000). Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. Journal of the American Medical Association 283, 2281–90.[Abstract/Free Full Text]

7 . Motin, V. L., Georgescu, A. M., Elliott, J. M. et al. (2002). Genetic variability of Yersinia pestis isolates as predicted by PCR-based IS100 genotyping and analysis of structural genes encoding glycerol-3-phosphate dehydrogenase (glpD). Journal of Bacteriology 184, 1019–27.[Abstract/Free Full Text]

8 . Comité de l’Antibiogramme de la Société Française de Microbiologie. (2003). Report 2003. International Journal of Antimicrobial Agents 21, 364–91.[CrossRef][ISI][Medline]

9 . Frean, J. A., Arntzen, L., Capper, T. et al. (1996). In vitro activities of 14 antibiotics against 100 human isolates of Yersinia pestis from a southern African plague focus. Antimicrobial Agents and Chemotherapy 40, 2646–7.[Abstract]

10 . Rahalison, L., Guiyoule, A., Bonacorsi, S. P. et al. (2000). Failure of oily chloramphenicol depot injection to treat plague in a murine model. Journal of Antimicrobial Chemotherapy 45, 541–5.[Abstract/Free Full Text]