Antibiotic-resistance Transfer in Yersinia enterocolitica BECHTEL, JR., M.D., AND JOHN R. BORING, III, PH.D.

REPORTED CASES of infection caused by Yersinia enterocolitica have increased over the past several years, indicating a more general awareness of this organism and perhaps an actual increase in incidence of the infection. 4,5,810 " 12 The clinical isolates of Y. enterocolitica have manifested few variations in their susceptibilities to antibiotics, most strains being sensitive to chloramphenicol, gentamicin, tetracycline, streptomycin, kanamycin, and sulfamethoxazole-trimethoprim. 4 ' 9 They are variably resistant to ampicillin and the cephalosporins. This general susceptibility may indicate that Y. enterocolitica does not acquire antibiotic resistance from other bacteria by the mechanism of gene transfer, or does so at a relatively low frequency. The apparent increase in the incidence of Y. enterocolitica infections may substantially increase the likelihood for these bacteria to come in contact with antibiotic-resistant organisms in nature. Thus, it seemed important to determine whether Y. enterocolitica had the potential to become antibiotic-resistant through contact with drug-resistant organisms. Kimura has reported transfer of single streptomycin resistance from one of three swine isolates of Y. enterocolitica to Escherichia coli at a frequency 4.1 x 10 -4 transconjugants (recipient cells that have acquired resistance) Received March 8, 1977; received revised manuscript and accepted for publication November 4, 1977. Address reprint requests to Dr. Boring: Department of Preventive Medicine, Emory University School of Medicine, 69 Butler Street, S.E., Atlanta, Georgia 30303. 0002-9173/79/0100/0093 $00.70 © Ai

Departments of Pathology and Preventive Medicine, Emory University Medical School, Atlanta, Georgia

per donor. 6 To our knowledge, transfer into and out of Y. enterocolitica has not been further reported in the English-language medical literature. The present study was undertaken to determine whether Y. enterocolitica could acquire resistance in vitro to antibiotics after exposure to other bacteria capable of transfer of resistance. Materials and Methods Bacterial

Strains

Escherichia coli 083:K untypable:H14 nalR (nalidixic-acid-resistant, MIC = 25 /xg/ml) was obtained from Dr. Dennis Schaberg at the Center for Disease Control in Atlanta, Georgia. Salmonella Indiana was isolated at Grady Memorial Hospital in Atlanta from a patient who had recently immigrated from Mexico. Yersinia enterocolitica strain Y40 was isolated from a patient at the same hospital. 7 Four additional strains of Y. enterocolitica (Yl, Y2, Y3, Y4) were obtained from Dr. Robert Weaver at the Center for Disease Control in Atlanta. Transfer of

Resistance

Trypticase SQy broth*, 20 ml, was inoculated from a single colony of either the donor culture or the recipient culture and incubated 18 to 24 hours at 37 C. These cultures, containing approximately 10 9 -10 10 bacteria per milliliter, were then mixed by adding 2 ml donor culture and 2 ml recipient culture to 16 ml trypticase soy broth. This resulted in a 1:1 mixture of organisms diluted 1:10. Three-to-one (dononrecipient) and 12:1 mixtures were prepared in a similar manner. Mixtures of donor and recipient cultures were incubated stationary at 37 C for 2 hours. One-tenth milliliter of the original donor culture, 0.1 ml of the recipient culture, and 0.1 ml of the mixture of donor and recipient cultures were each plated in duplicate onto MacConkey agar* containing single antibiotics for controls and two antibiotics for the selection of transcon* Bioquest, Division of Becton-Dickinson & Co., Cockeysville, Md. 21030. ican Society of Clinical Pathologists

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Bechtel, Jack, Jr., and Boring, John R., Ill: Antibioticresistance transfer in Yersinia enterocolitica. Am J Clin Pathol 71: 93-96, 1979. Most clinical isolates of Yersinia enterocolitica have been reported to be inhibited by a wide variety of antimicrobial agents. This finding indicates that Y. enterocolitica infrequently acquires resistance by gene transfer from other bacteria in nature. In the present study the authors examined whether Y. enterocolitica has the potential for acquiring antibiotic resistance from another organism in vitro. Mixed cultures of multiple-antibiotic-resistant Escherichia coli and a sensitive Y. enterocolitica yielded resistant Y. enterocolitica. Resistance to at least four drugs was transferred at a frequency of approximately 10~7 transconjugants per donor. The newly resistant Y. enterocolitica transferred resistance to a sensitive E. coli at a slightly higher frequency. Resistance to at least one antibiotic was transferred to all five strains of Y. enterocolitica tested. (Key words: Yersinia enterocolitica; Antibiotic resistance; Antibiotic-resistance transfer; Resistance plasmids.)

A.J.C.P. • January 1979

BECHTEL AND BORING

94

Table 1. Transfer Experiments

Donor (D) Experiment 1

Y. enlerocolitica (Y40) (Nals, Amp")

Experiment 2

S. indiana (Nals, TeR) R

Selective Drugs

Recipient R

s

s

Result (Designation)

Transfer Frequency (No./D/2 Hours) Less than 10"8

E. coli 083 (Nal , Amp )

Amp + Nal

No detectable transfer

E. coli 083 (NalR, Tes)

Te + Nal

Transfer: E. coli (R)

3 x 10-6

Experiment 3

E. coli (R) (Nal , Amp , TeR)

Y. enterocolitica (Y40) (Nals, AmpR, Tes)

Amp + Te

Transfer: Y. enterocolitica (Y40M)

3 x 10"7

Experiment 4

Y. enterocolitica (Y40M) (Nals, AmpR, TeR)

E. coli 083 (NalR, Amps, Tes)

Nal + Te

Transfer: E. coli (R-2)

6 x 10"6

S = sensitive; R - resistant. Nal = naladixic acid; Amp = ampicillin; Te = tetracycline.

Identification of Organisms Organisms were identified with the use of the modified R/B system,t incubated at 37 C. The tests employed were phenylalanine deamination, H2S formation, gas formation, lysine decarboxylation, indole formation, motility, ornithine decarboxylation, growth in citrate, DNAse production, and glucose, lactose, rhamnose, raffinose, sorbitol, and arabinose fermentations. In addition, motility and indole tests were carried out at 25 C. t Diagnostic Research, Inc. 25 Lumber Road, Roslyn, Long Island, N. Y. 11576.

Determination of Antibiotic Sensitivities Organisms that had acquired resistance were tested for antibiotic sensitivity at the same time as donor and recipient cultures to eliminate possible day-to-day variation. Antibiotic sensitivity testing was done on Mueller-Hinton agar with antibiotic disks and interpreted on the basis of the size of inhibition zones, with the use of the criteria of Bauer and associates.2 Antibiotics used and disk contents are indicated in Table 2. Minimum inhibitory concentrations (MIC) were determined by the tube dilution method, using a Mueller-Hinton broth containing graded concentrations of the antibiotic under test. The inoculum of organisms was approximately 105/ml. Results Yersinia enterocolitica Y40 incubated with the antibiotic-sensitive E. coli (ampicillin-sensitive and naladixic acid-resistant) did not give rise to lactose-positive colonies resistant to 10 /xg/ml ampicillin (Table 1, experiment 1), indicating failure of transfer at detectable frequency. The demonstration of transfer of tetracycline resistance from S. indiana to Y. enterocolitica proved difficult, in part because the recipient yersiniae were relatively antibiotic-sensitive to most drugs and were, like S. indiana, lactose-nonfermenting. It was thus decided to transfer resistance from S. indiana to sensitive E. coli and then to attempt to use E. coli (now resistant) as a donor for transfer of resistance to Y. enterocolitica, using as the unselected marker the failure to ferment lactose on the selective antibioticcontaining media. The mating between S. indiana and E. coli resulted in acquisition by the E. coli of resistance to four antibiotics: chloramphenicol, tetracycline, streptomycin, and sulfisoxazole (Table 1, experiment 2). Resistance to ampicillin and resistance to carbenicillin

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jugants. Antibiotics used were nalidixic acid (25 jug/ml) or tetracycline (10 jug/ml) for experiments involving E coli or S. indiana and ampicillin (10 /ug/ml) or tetracycline (10 /ug/ml) for experiments involving Y. enterocolitica (see Table 1 for a summary of antibiotics used in each experiment). Preliminary studies showed that these concentrations were insufficient to inhibit resistant strains and sufficient to inhibit all sensitive strains. Requirements for a valid experiment were growth of the donor organism on agar containing antibiotic " a " but not on agar containing antibiotic " b , " and growth of the recipient organism on " b " but not on "a." Transfer was considered to have occurred when lactosenegative or -positive colonies (depending on the experiment) grew on the plate containing both antibiotics " a " and " b . " For example, in a typical experiment involving transfer from resistant E. coli (resistant to nalidixic acid, tetracycline, and Other antimicrobial drugs) to sensitive Y. enterocolitica (sensitive to tetracycline and resistant to ampicillin), the three types of selective media would have been MacConkey agar with (1) ampicillin, (2) tetracycline, and (3) both ampicillin and tetracycline.

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RESISTANCE TRANSFER IN Y. ENTEROCOLITICA

Vol.71 . N o . I

Table 2. Antibiotic Sensitivity Patterns Antibiotic

c

s

(30)

(10)

G (250)

Amp (10)

Cr (30)

Sxt (25)

Nal

GM (10)

K (30)

CI (10)

St

S

s

s

S

S

S

R

S

s

S. indiana

R (125)*

R (250)

R

R

R

S

R

S

S

S

s s

E. coli (R)

R (250)

R (250)

R

R

S

S

S

R

S

S

s

S

S

s

S

R

R

s

S

s

S

s

R (125)

R (125)

R

R

R

R

s

S

s

S

s

enterocolitica (Yl)

I

S

S

S

S (6.25)

I

s

s

(Y2)

S

S

s

s

R (50)

R

s

s

(Y3)

S

S

s

s

I (6.25)

I

s

s

(Y4)

s

s

s

s

R (>50)

R

s

s

E. coli

Y.

enterocolitica (Y40)

Y.

enterocolitica (Y40M)

Y.

* Abbreviation of antibiotic. Number in parenthesis is disk content. Te = tetracycline; C = chloramphenicol; S = streptomycin; G = sulfisoxazole; Amp = ampicillin; Cr = cephalothin; Sxt = sulfamethoxazole + trimethoprim; Nal = naladixic acid; GM - gentamicin;

K = kanamycin; CI = colistin. t R = resistant; S = sensitive; 1 = intermediate. $ Minimum inhibitory concentration, pLg/ml.

apparently were not transferred. The newly resistant E. coli (R) was mated with Yersinia Y40, with the result that the latter organism acquired resistance (Table 1, experiment 3). The newly antibiotic-resistant Y. enterocolitica (designated Y40M) was mated with the sensitive E. coli to determine whether transfer of resistance could proceed in both directions. Transfer occurred (Table 1, experiment 4), thereby demonstrating that resistance to four antibiotics could be transferred from S. indiana to E. coli to Y. enterocolitica and back to E. coli. The sensitivity patterns to various antibiotics and the minimum inhibitory concentrations to tetracycline and chloramphenicol of various organisms examined are given in Table 2. Transconjugants were picked from selective plates and purified, and their antibiotic sensitivities and biochemical reactions were tested after each step in the process of transfer. Additional experiments to confirm transfer to other strains of Y. enterocolitica were performed with mixtures of resistant E. coli and the laboratory strains of Y. enterocolitica (Yl, Y2, Y3, and Y4). Strains Yl and Y3, but not Y2 and Y4, were sensitive to ampicillin. Resistance was acquired by all Y. entero-

colitica strains (Table 3). The donor E. coli used in these experiments was found to have lost resistance to chloramphenicol but remained resistant to tetracycline, streptomycin, and sulfisoxazole. The ampicillin-sensitive Y. enterocolitica (Yl and Y3) acquired resistance to tetracycline only. The biochemical reactions for Y. enterocolitica were typical. All strains were indole-negative at both 25 and 37 C, and were motile at 25 C but nonmotile at 37 C. Table 3. Resistance Patterns of Y. enterocolitica Following Mating with E. coli

Strain

Te

Sm

G

Amp

Cr

Transfer Frequency (No./Dt/2 Hours)

Y1M Y2M Y3M Y4M Y40M

Rt R R R R

S R S R R

S R S R R

S R S R R

I R I R R

3 6 6.9 2.7 8.1

Antibiotic

x 10-7 x 10-o x lQ-o x 10"8 x 10"'

* Te = tetracycline; Sm - streptomycin; G = sulfisoxazole; Amp = ampicillin; Cr ^ cephalothin. t D = donor. t R = resistant; S = sensitive.

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Te* (30)

Organism

96

BECHTEL A N D BORING

The Y. enterocolitica Y40M (with acquired resistance) was considered to grow more slowly than the parent organism, and some isolates of this organism required 72 hours at 25 C to develop motility. All the Yersinia strains grew more slowly on MacConkey agar than on Trypsticase soy agar at 37 C. Colonies on MacConkey agar were not well developed until after 48 hours at 37 C, a fact important in selecting bacteria that have acquired resistance. Discussion

The reason for this is not known, but it suggests that Y. enterocolitica is a poor recipient for plasmid transfer from other Enterobacteriaceae. The transfer of antibiotic resistance to Y. enterocolitica demonstrated in these studies indicates the potential for development of multiple-antibiotic resistance in nature. The relatively low frequency of transfer in vitro may indicate a similar low frequency in vivo and may be the reason that most clinical isolates are currently sensitive to many antibiotics. However, a greater incidence of infections with Y. enterocolitica may aliow an increased contact in nature between these organisms and multiply-resistant bacteria, with a resulting increase in resistant Y. enterocolitica. This implies the possibility for development of therapeutic problems, and both the clinician and laboratory personnel should be aware of the need to evaluate fully the antibiotic sensitivities of Y. enterocolitica isolates. References 1. Anderson ES, Lewis MJ: Drug resistance and its transfer in Salmonella typhimurium. Nature 206:579-583, 1965 2. Bauer AW, Kirby JC, Sherris JC et al: Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45:493-496, 1966 3. Grant RB, Bannatyne RM, Shapley AJ: Resistance to chloramphenicol and ampicillin of Salmonella typhimurium in Ontario, Canada. J Infect Dis 134:354-361, 1976 4. Gutman LT, Ottesen EA, Quan TJ, et al: An inter-familial outbreak of Yersinia enterocolitica enteritis. N Engl J Med 288:1372-1377, 1973 5. Keet E: Yersinia entercolitica septicemia. NY State J Med 74: . 2226-2229, 1969 6. Kimura S, Eda T, Ikeda T, et al: Detection of conjugative R plasmids in genus Yersinia. Jpn J Microbiol 19:449-451, 1975 7. Kohl S, Jacobson JA, Nahmias A, et al: Yersinia enterocolitica infections in children. J Pediatr 89:77-79, 1976 8. Nilehn B: Studies on Yersinia enterocolitica. Acta Pathol Microbiol Scand 206(suppl):l-48, 1969 9. Rabson AR, Hallett AF, Koornhof HJ: Generalized Yersinia enterocolitica infection. J Infect Dis 131:447-451, 1975 10. Schleifstein JI, Coleman MB: An unidentified microorganism resembling B. ligniere and Past, pseudotuberculosis, and pathogenic for man. NY State J Med 39:1749-1753, 1939 11. Toivanen P, Toivanen A, Olkkonen L, et al: Is the incidence of Yersinia enterocolitica infection increasing? Acta Pathol Microbiol Scand 82 (Section B):303-304, 1974 12. Toma S: Survey on the incidence of Yersinia enterocolitica in the Province of Ontario. Can J Public Health 64:477-487, 1973

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Transfer of resistance to chloramphenicol, tetracycline, streptomycin, and sulfisoxazole occurred from S. indiana to E. coli, and in turn from E. coli to Y. enterocolitica Y40. Since no biochemical reaction changed during testing procedures and naladixic-acid resistance was not transferred, chromosomal recombination is an unlikely explanation. The size of the genetic unit (four resistance determinants) makes transformation a highly unlikely mechanism. Transfer of resistance determinants in this study was probably via a resistance plasmid. The frequency of acquisition of resistance by Y. enterocolitica after exposure to resistant cells of E. coli for two hours was relatively low when compared with resistance transfers between many other organisms, such as E. xoli and Shigella. However, resistance pldsmids originating in Salmonella species have been shown to transfer at a somewhat low frequency. Grant has demonstrated that the frequencies of transfer of linked resistance to chloramphenicol, streptomycin, kanamycin, and tetracycline from several strains of Salmonella to E. coli ranged from 2 x 10-5 to 3 x 10~6 transconjugants per donor.3 Anderson demonstrated tetracycline-resistance transfer from Salmonella typhimurium to E. coli at a frequency of 2 x 10-6 transconjugants per donor after overnight incubation of mixtures.1 Our results indicate a similar transfer frequency from Salmonella indiana to E. coli, and from Y. enterocolitica to E. coli. The number of transconjugants obtained by mating resistant E. coli with sensitive Y. enterocolitica was generally fewer than in the reciprocal cross.

A.J.C.P. . January 1979

Antibiotic-resistance transfer in Yersinia enterocolitica.

Antibiotic-resistance Transfer in Yersinia enterocolitica BECHTEL, JR., M.D., AND JOHN R. BORING, III, PH.D. REPORTED CASES of infection caused by Ye...
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