World

Journal

of Microbiology

and Biotechnology,

9, 365366

Short Communication

Anti-microbial susceptibility of YeWM pseudotuberculosis and Yersinia enterocoh’tica under different cultural conditions N. Markova,* T. Radoucheva, L. iiieva and D. Veljanov Generally, YersEa pseudoUer&osis antibiotics than when grown at X°C. phenicol of Yersjnja was also influenced

and Y. enterocoMca grown at 3T°C had The susceptibility to kanamycin, cephalothin, by the growth medium and gas composition.

fiy

susceptibility,

words; Antibiotics,

cultural

conditions,

increased susceptibility to tetracycline and chloram-

Yersinia.

The anti-microbial susceptibility of Yersiniu pseudotuhmlosis and Yersinia enterocolifica becomes important when they are naturally transformed from the environment to their hosts. The temperature-regulated surface properties of Yersinia spp. are associated with the synthesis of different surface structures (proteins, lipopolysaccharide) from the outer membrane (Acker et cd. 1980; Skumik 1985; Gronberg & Kalstrom 1989). Kouwatli ef al. (1979) suggested that the temperature of incubation directly influenced the antimicrobial susceptibility of Yersinia. The purpose of the present study was to investigate the anti-microbial susceptibility of Y. pseudofuberculosis and Y. enterocolifica grown in different cultural conditions which modelled environmental and host body milieu.

Anfibiofic Sensitioify Tesfing Antibiotics (kanamycin, erythromycin, cephalothin, chloramphenicol, ampicillin and tetracycline) were obtained commercially. For minimum inhibiting concentration (MIC) assays, bacteria were suspended in meat-peptone broth to provide inocula containing approximately I.2 x IO6 cells/ml (detemined optically). Serial two-fold dilutions of the antibiotics were prepared in meat-peptone broth and distributed in glass tubes; each tube contained 1.0 ml of an antibiotic dilution and 1.0 ml of inoculum. Tubes were incubated at 37 or 25’C for 24 h. The MIC values given are the lowest antibiotic concentrations that gave complete inhibition of visible growth. For each antibiotic, three experiments were performed, each with three replicates.

Materials

Results

and Methods

Bacferia and Grouh Corzditions Yminia pseudofuberculosis serovar III and Yershia mferoco/ifica 0: 3 were a kind gift from Prof H. MoIlaret of the International Yersinia Centre, Paris. Eight different culture-condition combinations were constructed involving two growth media [meat-peptoneiagar or Hanks solution with (g/l): NaCI, 80.0; KCI, 4.0; MgSOd.7H20, 2.0; CaC12, 1.4; KHzP04, 0.6; Na2HPO+ZH20, 0.6; glucose, IO; Phenol Red, 1.0; pH 7.2, two temperatures (ZS’C and 37°C) and

N. Markova, T. Radoucheva, of Microbiology, Bulgarian *Corresponding author @I 1993 Rapid

Communications

L. llieva and D. Veljanov are with the Institute Academy of Sciences, 1113 Sofia, Bulgaria.

of Oxford

two gas compositions [air or air with were grown for 24 h.

10% (v/v)

COJ.

The bacteria

and Discussion

The susceptibilities to antibiotics of Y. pseudofuberculosis III and Y. ewterocoliticu 03 are summarized in Table 1. Yersinia pseudofuberculosis and Y. enferocolifica grown at 57’C in Hanks solution had the highest susceptibilities to kanamycin and tetracycline. Cultural conditions had no significant effect on susceptibility of either species to erythromycin and ampicillin. At 3VC Yersinia mterocolifica grown in Hanks solution was always more resistant to cephalothin than when grown in a meat-peptonelagar. Yersinia pseudotuberculosis grown at 25C had MIC values for cephalothin

Ltd World ]ourmf

of

Microbiology

and Biotechnology, Vol 9, 1993

385

N. Markova

Table grown

et

al.

1. MinImal inhibitory concentrations under different cultural conditions. Temperature

Medium

Gas’

of antlbiotlcs

Kanamycin

@g/ml)

against

Yers/n/a

Cephaiothin

enfemco//fke

Erythromycin

and

Yershle

Tetracycline

pseudofubercidosis

Chloramphenicol

WI MPAt MPA

25

Air

25

Air

MPA MPA

37

Hank’s Hank’s

25 25

Hank’s Hank’s

37 37

MPA MPA

25

MPA

37 37

37

Hank’s Hank’s

25

Hank’s

37 37

64

4 * 0.66*

4 * 0.29

+ co2 Air Air

6 k 0.66

32 k 6.1$ 32 k 5.62s

64 64

4 * 0.70* 4 * 0.66$

4 * 0.29

6 k 0.56

+ co2 Air

6 k 0.56 6 k 0.66

32 k 5.62$ 64 k 10.5

64

4 * 0.66$

64

6 k 0.66

2 * 0.33 4 * 0.33

6 k 0.66

126 k 15.14$

64

6 k 0.70

2 * 0.33

126 * 15.14$

64

2 * 0.33$

+ co* Air Air

2 * 0.35 4 * 0.33*

64 & 11.24 0.5 k 0.06$

64 126

2 * 0.35* 4 * 0.35%

2 * 0.33

+ co* Air

4 * 0.33* 2 k 0.16

0.5 k 0.06

126

4 * 0.33$

1 k 0.06

126

4 * 0.35$

6 * 0.66 6 & 0.70

2 * 0.17 2 & 0.33

1 k 0.067 0.5 k 0.063$

126 256

2 * 0.35 2 k 0.16

4 * 0.70

2 & 0.33

+ co2 Air

used

at 10%

7 MPA-Meat-peptonelagar. $ Significantly different All MIC

values

for

+ co2 Air Air + co2 Air

6 k 0.56

4 * 0.29

0.5 k 0.063%

126

0.5 k 0.06

1 k 0.067

126

2 & 0.16 1 k 0.16$

4 * 0.29 2 * 0.33

0.5 & 0.06

1 * 0.067

126

1 * 0.17$

2 * 0.29

Air

(v/v)

in air.

(P < 0.05)

ampicillin

were

from

one

doubling

dilution,

126 mg/ml.

lower than those obtained at SW. In contrast, the chloramphenicol MIC for Y. pseudotuberculosis and Y. enterocolitica were generally higher at x’C than at 3i”C. It is possible that the combination of Hank’s growth medium with CO2 at 3i”C, which mimicks the host body milieu, leads to resistance against antibiotics with clinical imporL3nce. These results suggest that variation in environmental conditions affects Yersinia spp. susceptibility to antibiotics-a property, known as phenotypic tolerance, which occurs frequently in response to changes in the environment and is reversible (Tuomanen et al., 1986). The altered susceptibility of Yersinia is possibly at the level of antibiotic interaction with the cell surface. The outer membrane permeability of each Yersinia bacterium is closely related to its cell wall composition, and this is dependant on environmental factors. Therefore, the variation in response of Yersinia to antibiotics under different cultural conditions may be the result of variation in the diffusion and penetration of the tested antibiotics into the bacterial cell. This may be reflected in changes in the ability of the

386

4 * 0.33 2 * 0.33

Air

+ co* * CO2 was

4 * 0.33

Air

25

Hank’s

64 k 5.3

Air

25

MPA

6 & 0.66

World Jownal of Microbiology md Biotechdqy,

Vd 9, 1993

bacteria to resist the bactericidal effect of antibiotics the natural epidemiological hot-cold cycle.

during

References Acker, G., Wartenberg, K. & Knapp, W. 1980 Sugar composition of the lipopolysaccharide and ultrastructural study of the outer membrane of Yersiniu enhxohh, Zentru/bI& )i2r Bukteriologie und Hygiene, 1 AbteiLng Originul A 249, ZZ9-XO. Gronberg, A. & Kalstrom, E. 1989 Structural variations and growth potential of Y. mter&iticu under different culture conditions. A& Puthologicu, Ivficrobio/ogicu et hnmwologicu Scandinavicu 97, 227-235. Kouwatli, K., Bejar Ruiz-Bravo, A. & Ramos-Cormenzana, A. 1979 Sensitivity of Y. enterocoliticu to several antimicrobial agents. &ficrobios Letters 11, 137-142. Skumik, M. 1985 Expression of antigen encoded by the virulence plasmid of Y. enterocoliticu under different growth conditions. InfecGon und Immunity 47, 183-190. Tuomanen, E., Durack, D.T. & Tomasz, A. 1986 Antibiotic tolerance among clinical isolates of bacteria. Antitnicrobia! Agents and Chemotherapy 30, 521-527. (Received

in revised

accepted

27 November

form

23 November 1992)

1992;