Comp. lmmun. Microbiol. infect. Dis. Vol. 12, No. 1/2, pp. 5-8, 1989 0147-9571/89 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1989 Pergamon Press pie

IN VITRO SUSCEPTIBILITY OF P S E U D O M O N A S MALLEI TO ANTIMICROBIAL

AGENTS

S. A. AL-IzzI and L. S. AL-BASSAM

Department of Medicine, College of Veterinary Medicine, A1-Ameria, Baghdad, Iraq

(Received 21 June 1988)

Almtraet--Pseudomonas mallei was isolated from pus samples obtained from 34 mallein-positive horses. The isolates were subjected to in vitro sensitivity test using 16 different antimicrobial discs. All isolates (34) were sensitive to sulfamethizole, gentamycin, tetracycline, sulfathiazole, kana- mycin, tobramycin, streptomycin and a combination of trimethoprim and sulfamethoxazole while none of them were sensitive to cephalothin, colistin, ampicillin, penicillin and nitrofurantoin. Rifapicin, chloramphenicol and carbenicillin were effective against 32, 26 and 18 isolates respectively. The minimum inhibitory concentrations (MICs) of gentamycin, tetracycline, to- bramycin, sulfamethizole, streptomycin, rifampicin and a combination of trimethoprim and sulfamethoxazole were 0.28, 0.38, 0.67, 1.40, 3.40, 5.86 and 5.30/~g/ml, respectively.

Key words: Antibio susceptibility, Pseudomonas mallei, antimicrobial agents.

Rrsumr---Pseudomonas mallei a dt6 isol6 de pus provenant de 34 chevaux positifs ~. la mallrination. L'antibiogramme des souches isolres a 6t6 rralisd avec 16 antibiotiques diffrrents (mrthode des disques). Toutes les souches se sont rrvrlres sensibles ~i sulfamethizole, gentamicine, t&ra- cycline, sulfathiazole, kanamycine, tobramycine, streptomycine et rassociation tdmrthoprim- sulfamethoxazole tandis qu'aucune n'rtait sensible fi cephalothine, colistine, apicilline, penicilline et nitrofurantoine. La rifapicine, le choloramphenicol et la carbenecilline 6taient actives sur respectivement 32, 26 et souches.

Mots-clefs: antibiosensibilitr, Pseudomonas mallei, antibiotique.

I N T R O D U C T I O N

Glanders is an infectious disease caused by Pseudomonas mallei. It is primarily a disease of solipeds which can also affect other animals particularly those which have fed on infected horse meat [1-3]. The infection in man is usually fatal [4]. Glanders has been almost completely eradicated from many countries such as United States and Canada [5] while it is still prevalent in others such as Turkey [1], India [6] and Iraq [7].

There are few studies on the sensitivity of P. mallei to antimicrobial agents [8-10]. The knowledge of the in vitro activity of various drugs against P. mallei strains is necessary for treating human patients infected with this micoorganism. For this reason, the in vitro susceptibility of 34 P. mallei isolates to 16 different antimicrobial agents was determined.

M A T E R I A L S A N D M E T H O D S

Pseudomonas mallei was isolated from pus samples obtained from nodules on the skin of 34 mallein-positive horses. The isolates were identified on the basis of their cultural,

6 S . A . AL- Izz t and L. S. AL-BASSAM

morphological and biochemical characteristics as suggested by Cown and Steel [11]. The colonies of all isolates were small yellowish grey with smooth glistening convex surfaces on brain and heart infusion agar after 48 hr of incubation at 37°C. They did not grow at 42°C and on MacConkey's agar. Gram's stained smears prepared from 48 hr old cultures revealed gram negative long rods. All isolates were non motil, oxidase positive and failed to hydrolyse starch, casein and gelatin. None of these isolates produced H2 S on triple sugar iron agar or decomposed urea. The isolates reduced nitrate, utilize glucose oxidatively but failed to decarboxylize lysine and to utilize citrate. Strauss reaction was observed on guinea pigs inoculated with these isolates.

Antibiotic sensitivity test was performed according to Bauer et al. [12] using 16 different antimicrobial discs. The following bioMerieux discs were used: sulfamethizole, gen- tamycin, tetracycline, sulfathiazole, kanamycin, tobramycin, streptomycin, rifampicin, chloramphenicol, carbenicillin, cephalothin, colistin, ampicillin, penicillin G, nitro- furantoin and a combination of trimethoprim and sulfamethoxazole (BioMerieux, Char- bonnieres les Bains, France).

The MICs of gentamycin, tetracycline, tobramycin, sulfamethizole, streptomycin, rifampicin and a combination of trimethoprim and sulfamethoxazole (1:19) were deter- mined using agar dilution method as described by Washington and Barry [13]. Preparation of inoculum was made by emulsifying 4-5 colonies from 48 hr culture of P. mallei into 4 ml of trypticase soy broth and adjusted to the turbidity of the barium sulfate standard. This turbidity was equivalent to approximately 1 x 108 to 5 x l0 s colony forming units. A !:20 dilution was prepared in Mueller-Hinton broth then 0.002 ml was inoculated on Mueller-Hinton agar containing antimicrobial agents. The MIC was read after overnight incubation at 37c'C as the lowest concentration of antimicrobial agent at which complete inhibition occurred.

RESULTS

Results of in ~'itro sensitivity of P. mallei isolates to antimicrobial agents are shown in Table 1. The patterns of susceptibility of the 34 isolates examined in this study were almost uniform. The most effective antimicrobial agents against these isolates were sulfamethizole,

Table 1. In vitro sensitivity of 34 Pseudomonas mallei isolates to antimicrobial agents

No. of Pseudomonas isolates Antimicrobial agent Disc contents Sensitive Intermediate Resistant

- Sulfamethizole 1 mg 34 0 0 Gentamycin I 0 mcg 34 0 0 Tetracycline 30 mcg 34 0 0 Sulfathiazole 1 mg 34 0 0 Kanamycin 30 mcg 34 0 0 Tobramycin 30 mcg 34 0 0 Streptomycin 10 mcg 34 0 0 Trimethoprim + sulfamethoxazole 1.25 + 23.75 mcg 34 0 0 Rifampicin 30 mcg 32 0 2 Chloramphenicol 30 mcg 20 6 8 Carbenicillin 50 mcg 17 1 16 Cephalothin 30 mcg 0 0 34 Colistin 10 mcg 0 0 34 Ampicillin 10 mcg 0 0 34 Penicillin G l0 IU 0 0 34 Nitrofurantoin 300 mcg 0 0 34

Pseudomonas mallei: suscept ibi l i ty to an t imic rob ia l agents

Table 2. Means and ranges of minimal inhibitory concentrations of antimicrobial agents

Minimum inhibitory concentration (~g/ml)

Antimicrobial agent Mean Range

Gentamycin 0.28 0.1 0.4 Tetracycline 0.38 0.2~).4 Tobramycin 0.67 0.4~).8 Sulfamethizole 1.40 0.8 1.6 Streptomycin 3.40 3.2 6.4 Rifampicin 5,86 3.2 6.4 Trimethoprim + sulfamethoxazole (1 : 19) 5,30 3.2 12.5

gentamycin, tetracycline, sulfathiazole, kanamycin, tobramycin, streptomycin and a combination of trimethoprim and sulfamethoxazole. The MICs of gentamycin, tetra- cycline, tobramycin, sulfamethizole, streptomycin, rifampicin and a combination of trimethoprim and sulfamethoxazole are shown in Table 2. Of the drugs tested, gentamycin was the most active, with a mean MIC of 0.28/~g/ml.

DISCUSSION

Glanders was diagnosed on the basis of mallein test and the isolation of P. mallei in 34 horses. These horses were destroyed because they can act as a source of infection to other animals and man especially stable men, owners and veterinarians. Clinical cases of glanders have occurred among horse owners [4] and laboratory technicians [1] and more cases probably will be diagnosed in areas where the disease is still endemic, and require therapy. Determination of the susceptibility of P. mallei isolates to antimicrobial agents is a key for treating glanders.

Antibiogram revealed that all P. mallei isolates were sensitive to sulfamethizole, gentamycin, tetracycline, sulfathiazole, kanamycin, tobramycin, streptomycin and a combination of trimethoprim and sulfamethoxazole while most of them were sensitive to rifampicin and chloramphenicol. These findings were in general agreement with those of other investigators [8, 9] who found that P. mallei were sensitive to tetracycline, kana- mycine, streptomycin and sulfonamides. Similar activities of gentamycin and tobramycin against P. aeruginosa [14] and chloramphenicol and a combination of trimethoprim and sulfamethoxazole but not rifampicin against P. pseudomallei [15, 16] were observed. Resistance of P. mallei isolates, examined here, to cephalothin, colistin, ampicillin and penicillin G was also encountered with P. pseudomallei [16].

The results of disc and plate dilution tests with gentamycin, tetracycline, tobramycin, sulfamethizole, streptomycin, rifampicin and a combination of trimethoprim and sul- famethoxazole correlated well and indicated that all isolates were within the sensitive range.

The susceptibility of P. mallei isolates to most antimicrobial agents used in this study was probably due to the fact that infected horses must not be treated but rather destroyed according to the rules. On the basis of in vitro antimicrobial sensitivity tests, gentamycin is a drug of choice for treating glanders.

Acknowledgement--The au thors t h a n k the staff o f cl inical pa tho logy for their technical assistance.

8 S.A. AL-IZzI and L. S. AL-BASSAM

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