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Original Article
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Volume 337:677-681 September 4, 1997 Number 10
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Multidrug Resistance in Yersinia pestis Mediated by a Transferable Plasmid
Marc Galimand, Ph.D., Annie Guiyoule, Guy Gerbaud, Bruno Rasoamanana, M.D., Suzanne Chanteau, Ph.D., Elisabeth Carniel, M.D., Ph.D., and Patrice Courvalin, M.D.

 

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Yersinia pestis is the causative agent of plague, a zoonotic disease transmitted to humans through flea bites and typically characterized by the appearance of a tender and swollen lymph node, the bubo. Human-to-human transmission can occur, through either the bite of fleas (bubonic plague) or respiratory droplets, causing an overwhelming infection called pneumonic plague.

The last plague pandemic began in Hong Kong in 1894 and spread throughout the world, establishing many endemic foci. Antibiotics and enforcement of public health measures significantly decreased the morbidity and mortality associated with the disease but did not allow its eradication. In fact, plague is now considered a reemerging disease1 for at least three reasons. First, there has been an increase in the number of cases reported to the World Health Organization.2 Second, plague reappeared in 1994 in an epidemic form in countries, including Malawi, Mozambique, and India, where it had been silent for 15 to 30 years. Third, the number of foci is gradually expanding in certain countries. In the United States for instance, the number of states reporting cases of human plague increased from 3 in the 1950s to 13 in the 1990s.3

Streptomycin, chloramphenicol, and tetracycline are used to treat plague, and tetracycline and sulfonamides are recommended for prophylaxis.4 Classically, Y. pestis isolates are uniformly susceptible to the antibiotics active against gram-negative bacteria.5,6,7

We report high-level resistance to multiple antibiotics, including all the drugs recommended for plague prophylaxis and therapy, in a clinical isolate of Y. pestis. The resistance genes were carried by a plasmid that could conjugate to other Y. pestis isolates. This report should serve as a warning of the risk of the spread of resistance in Y. pestis, a species previously considered universally susceptible to antibiotics.

Methods

Patient and Strains

The properties of the strains used are listed in Table 1. Y. pestis 17/95 biotype orientalis was isolated in 1995 in the Ambalavao district of Madagascar from a 16-year-old boy.5 The patient presented with fever, chills, and myalgia suggestive of malaria and was treated with quinine. Three days later, the appearance of a right inguinal bubo with high-grade fever (temperature, 41°C), delirium, and prostration led to the diagnosis of plague. The bubo was punctured, and the patient was treated with twice-daily intramuscular injections of streptomycin (2 g per day for 4 days) and oral trimethoprim–sulfamethoxazole (2 g per day for 10 days). The patient recovered but had severe asthenia for more than a month.

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  		Table 1. Properties of the Bacterial Strains Studied.

 
Mediums and Resistance Studies

Brain–heart infusion broth and agar (Difco) were used. The minimal inhibitory concentrations of antibiotics were determined on Mueller–Hinton agar (Sanofi Diagnostics Pasteur). The cultures were incubated for 48 hours at 28°C for Y. pestis and for 18 hours at 37°C for Escherichia coli. Chloramphenicol acetyltransferase and aminoglycoside-modifying enzymes were assayed in supernatants (centrifuged at 100,000xg) after ultrasonic disintegration.12,13 Matting on filters was performed as described previously.14

Nucleic-Acid Techniques

Isolation of plasmid DNA, cleavage of restriction fragments, and purification of DNA fragments from agarose type VII (Sigma Chemical) were performed as described elsewhere.9 Purified DNA fragments to be used as probes were labeled with [{alpha}-32P]deoxycytidine triphosphate by nick translation. Hybridization was carried out under highly stringent conditions.9 The polymerase chain reaction (PCR) was performed on a DNA thermal cycler (model 2400, Perkin-Elmer Cetus). Double-stranded DNA sequencing was performed by the dideoxynucleotide chain-termination method15 with a modified T7 DNA polymerase and [{alpha}-35S]deoxyadenosine triphosphate.

Results

Antibiotic Resistance of Y. pestis 17/95

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 3''-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

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

 
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 1.5x10-2 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.

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).


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  		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 tet(D) PCR product (Panel B). Lane 5 shows fragments obtained by the digestion of bacteriophage lambda DNA with HindIII and used as standards for molecular size.

 
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 x10-2. 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 x 10-4 and 5.7 x 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

Partial sequencing of an 864-bp PCR product obtained with oligodeoxynucleotides specific for blaTEM-1 indicated the presence of a cytosine at position 317 (numbering according to Sutcliffe19), confirming that resistance to ampicillin was due to the presence of the gene for TEM-1 penicillinase. The probe corresponding to catI hybridized to pIP1202 DNA, and the plasmid conferred to E. coli DB10 resistance to fusidic acid,20 indicating that resistance to chloramphenicol was due to the production of a type I chloramphenicol acetyltransferase. Probes internal to the aph(3')-I and aad(3'')(9) genes hybridized to pIP1202 DNA, confirming that resistance to kanamycin was due to the synthesis of a type I 3'-aminoglycoside phosphotransferase and that resistance to streptomycin–spectinomycin was due to the production of a 3''-9-aminoglycoside adenylyltransferase (data not shown). The tet (D) gene was detected by hybridization (Figure 1B), PCR, and sequencing of 520 internal base pairs. PCR amplification and sequencing of a 488-bp fragment internal to the sulI determinant confirmed that resistance to sulfonamide was due to the production of a drug-resistant dihydropteroate synthase. The sulI gene is nearly always located in conserved segments of integrons in Tn21-like elements that are carried by large conjugative plasmids.21 We used specific primers to amplify fragments internal to the integrase gene and the truncated ORFIV from Tn21-like elements from pIP1202 DNA by PCR, and the two genes were found to flank aad(3'')(9) alone.

Discussion

Y. pestis is considered universally susceptible to antibiotics recommended and widely used for prophylaxis and treatment of plague.4 In recent studies the isolates tested were susceptible in vitro to all antibiotics active against gram-negative bacteria,6,7 with the exception of tetracycline in rare cases.22

Multidrug-resistant Y. pestis 17/95 was isolated in 1995 in the Ambalavao district of Madagascar from a patient who presented with symptoms of bubonic plague.5 Despite extensive surveillance of strains of Y. pestis isolated between 1926 and 1995 in Madagascar, no multidrug-resistant strain was detected.5,22 Strain 17/95 was resistant not only to all the antibiotics recommended for therapy (chloramphenicol, streptomycin, and tetracycline) and prophylaxis (sulfonamides and tetracycline) of plague4 but also to drugs that may represent alternatives to classic therapy, such as ampicillin, kanamycin, spectinomycin, and minocycline. The isolate remained susceptible to cephalosporins, other aminoglycosides, quinolones, and trimethoprim, and treatment with trimethoprim, despite its lack of synergism with sulfonamides, most likely led to the patient's recovery.

The resistance determinants were carried by the conjugative plasmid pIP1202. Several observations strongly argue for an origin of this plasmid in a member of the Enterobacteriaceae family. The resistance genes carried by pIP1202 were closely related in structure to plasmid-borne determinants commonly found in enterobacteria. The Inc6-C origin of replication of pIP1202 was typical of plasmids of this group of bacteria with a broad range of hosts, and the plasmid was easily transferred in vitro from E. coli to Y. pestis.

The site of the putative genetic transfer remains unknown. Enterobacteria are usually confined to the intestinal lumen of the host, whereas Y. pestis circulates in lymphatic vessels, the spleen, the liver, blood, and sometimes the lungs. However, intestinal enterobacteria and Y. pestis may come into contact when gut bacteria invade the bloodstream. Alternatively, if pIP1202 originated in an invasive pathogen, the contact between the two microorganisms may have occurred in the blood or in deep tissues. A third possibility is that intimate contact between Y. pestis and the donor was achieved outside the mammalian host, perhaps in the gut of a flea that ingested blood infected with both microorganisms.

The fact that the multidrug-resistant plasmid was highly transferable in vitro to other strains of Y. pestis, where it was stable, is of great concern. It is likely that this type of replicon can also be transferred among strains of Y. pestis in their natural environment and, therefore, that resistance may spread locally in this species. Even more alarming, the observation that Y. pestis is able to acquire, under natural conditions, a resistance plasmid, regardless of its true origin, indicates that such a clinically ominous event may occur again.

Supported in part by a grant from Actions Concertées du Réseau International des Instituts Pasteur et Instituts Associés and by the Madagascar Ministry of Health.

We are indebted to Carmen Buchrieser for performing the pulsed-field gel electrophoresis.


Source Information

From the National Reference Center for Antibiotics and the Unité des Agents Antibactériens (M.G., G.G., P.C.) and the National Reference Laboratory–World Health Organization Collaborating Center for Yersinia (A.G., E.C.), Institut Pasteur, Paris; and the Plague Central Laboratory, Institut Pasteur, Antananarivo, Madagascar (B.R., S.C.).

Address reprint requests to Dr. Carniel at the National Reference Laboratory–WHO Collaborating Center for Yersinia, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris CEDEX 15, France.

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