Susceptibilities of Motile Aeromonas sp. to Antimicrobial Agents

Zbl. Bakt . 275, 85-93 (1991) © Gustav Fischer Verlag, StuttgartlNew York

Susceptibilities of Motile Aeromonas sp. to Antimicrobial Agents ADAM KAZNOWSKI and KRYSTYNA WLODARCZAK Department of Micob iology, Adam Mickiewicz University, Poznan, Poland

Received December 20, 1989 . Received in revised form August 10, 1990 . Accepted October 26, 1990

Summary Resistance to antimicrobial agents of 106 isolates of motile Aeromonas sp. was characterized . The results indicated that in vitro susceptibilities among the three species of the motile Aeromonas sp. were similar , and only the distribution of susceptibility to cephalotin was different. The percentage of resistance of A. sobria strains was lower than the percentage of the resistant strains of A . hydrophile and A . cauiae. All of the isolates were susceptible to kanamycin, nalidixic acid, tobramycin, amikacin, netilmicin, cefuroxime, ceftriaxone, cefoperazone and cefotaxime. Three of the tested stra ins (two A. hydrophila and one A. caviae) transferred resistance plasmids to the Aeromonas hydrophila recipient .

Zusammenfassung Die Resistenz gegen antibakterielle Substanzen wurde an 106 Stammen von beweglichen

Aeromonas spp . bestimmt. Die in vitro-EmpfindIichkeit der drei Species beweglicher

Aeromonaden war ahnlich, ausgenommen gegen Cephalotin. Der Prozentsatz resistenter Stamrne gegen Cephalotin war niedriger fur A. hydrophila und A. caviae. Aile 106 Starnme sprachen in vitro auf Kanam ycin, Tobramycin, Netilmicin, Amikacin, Nalidixinsaure sowie auf Cefuroxim, Cefotaxim, Ceftriaxon und Cefoperazon an. Zwei Starnme von A. bydrophila und ein Stamm von A. cauiae konnten die Resistenzplasmide auf A. hydrophila als Rezipienten iibertragen.

Introduction Motile Aeromonas sp . are Gram-negative bacteria, usually found in natural waters and sewage (31, 36). In the IXth edition of Bergey's manual, three motile species are recognized: A. hydrophila, A. caviae and A. sobria (31). Recently, four new species have been proposed: A. media (1), A. veronii (16), A. schubertii (17) and A. eucreno-

phila (37).

86

A. Kaznowski and K.Wtodarczak

Motile Aeromonas sp. may cause enteric and extraintestinal infections in humans (13, 18, 21, 42). A. hydrophila has been described as causing haemorrhagic septicaemia in a variety of fish species (3). The susceptibility of motile Aeromonas to antimicrobial agents has been found to vary, but most strains have usually been susceptible to new betalactams, aminoglycosides, chloramphenicol, tetracyclines, 4-quinoline derivatives and trimethoprim-sulfamethoxazole (9, 11, 12, 21, 23, 35). There are some differences between motile Aeromonas strains of human origin and strains of environmental origin. Some authors have frequently isolated strains resistant to chloramphenicol, streptomycin and tetracycline from water and fish (2, 22, 26, 32, 39). Some of the strains could transfer resistance determinants to E. coli andlor to S. typhimurium (15, 22, 32, 39). Strains of the Aeromonas sp. possessing R plasmids have also been detected in human specimens (15). McNicol et al. (26) and Hedges et al. (15) have isolated plasm ids from Aeromonas strains and have determined their size. Hedges et al. (15) found that some plasmids have been transmissible to E. coli, but as they are unstable in this recipient, the transfer of these plasmids to A. hydrophila failed. In the present study, susceptibility to antimicrobial agents of motile Aeromonas strains was determined. The strains of Aeromonas sp. resistant to antibiotics were examined for the presence of plasmids, and conjugation transfer.

Material and Methods

Bacterial strains. One hundred six bacterial strains of motile Aeromonas sp. were examined. The strains were isolated from drinking and surface water, municipal sewage, water-oil emulsion (aluminium rolling coolant) and fish (Ruti/us ruti/us). These strains were analysed by numerical taxonomy and their characters have been described previously (20). Sensitivity testing. The susceptibility of bacterial strains to 8 antibiotics was determined by a standard agar dilution procedure, using a Mueller-Hinton agar medium and an inoculum of 5 x 104 c.f.u. per spot (29). The MIC values were determined for the following antibiotics: ampicillin, chloramphenicol, gentamicin, kanamycin, colistin, streptomycin, tetracycline and nalidixic acid. The MIC breakpoints for susceptible and resistant categories were determined by NCCLS MIC interpretive standards (29). The activity of the following agents was tested against all strains by the disk method: tobramycin, carbenicillin" azlocillin, amikacin, netilmicin, cephalotin, cefuroxime, cefamandole, ceftriaxone, cefoperazone, cefotaxime, cefradine, rrimethoprim-sulfamethoxazole (bio-Merieux). Testing was done according to the disk manufacturer's instructions, results were intepreted using NCCL criteria (28). For sensitivity controlling tests, two reference strains were included: Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 (28,29). Resistance transfer experiments. The following strains were used as recil?1ents : E. coli K12 J53 [r, pro", met", RifR], E. coli K12 J62 [r, lac-, pro", trp", Nal ] (6) and A. hydrophi/a ATCC 7966 [AMpR, CARR, NaiR or AMpR, CARR, RifR] . A. hydrophila recipients were obtained after UV mutation . These strains were not used as recipients in examination transfer of ampicillin and carbenicillin resistance from donors. The technique for plasmid transfer was as described by Porazikoua et al. (32). The transfer method described by Thomas (40) was used when resistance to an antibiotic had been detected by the disk method. Transfer experiments were conducted at 30°C and 37°C.

Susceptibilities of Motile Aeromonas sp.

to

Antimicrobial Agents

87

Plasmid stability assay. Well-separated colonies on a Mueller-Hinton agar plate containing an antibiotic selective for bacteria carryi~ the plasmid were suspended in 1 ml Penassay broth. The suspension were then diluted 10 in Penassay broth and incubated overnight at 30°C and 3rC, respectively. The dilution and incubation steps were repeated 14 times. At intervals during the growth period, appropriate dilutions were plated directly onto nonselective Mueller-Hinton agar and the percentage of resistant colonies were determined by replica plating (8). Plasmid isolation and molecular weight determination. Plasmids were isolated by the method of Kado and Liu (19). Molecular weight was determined by horizontal electrophoresis in 0.7% and 1% agarose gel. The following reference strains possessing plasmids were used: E. coli J53-2 [R6K- 26 Md (7)], E. coli J53-2 [RP4- 38 Md (41)]; E. coli J53-2 [R6 - 65 Md (38)]; E. coli J53-2 [R27=TP117 -112 Md (14)]; E. coli P678-54 [col E1- 4.2 Md (8)]; E. coli RR1 [pBR322 - 2.6 Md (4)]; E. coli V 517 [35,8; 4.8; 3.7; 3.4; 2.6; 2.0; 1.8; 1.4 Md] (24).

Results and Discussion The distribution of MIC results of the 106 strains of motile Aeromonas spp. as determ ined by the agar dilution method has been listed in Table 1, and the susceptibil ity results obtained by the disk method are shown in Table 2. All of the strains of motile Aeromonas sp. were susceptible to kanamycin, nalidixic acid, tobramycin, amikacin, netilmicin and cephalosporines: cefuroxime, ceftriaxone, cefoperazone and cefotaxime . A high percentage of the strains was susceptible to colistin, gentamicin and azlocillin. These results are close to the results noted by other authors (10, 12,25,27,30). Only 2 of the strains were susceptible to ampicillin . Sensitivity of Aeromonas sp. to this antibiotic is noted rarely (34) whereas resistance is widespread (10, 12,26,32). Few strains were resistant to tetracycline, chloramphenicol and streptomycin. The resistance to these antibiotics of environmental and fish strains of motile Aeromonas spp. has also been reported previously (2, 26, 32, 39). Contrary to these results, Aeromonas strains of human origin are usually susceptible to these agents (11, 27, 34 ). Some tested Aeromonas strains were resistant to trimethoprim-sulfamethoxazole: A . hydrophila 22.6 %; A . caviae 37 .9%; A . sobria 40.3%, although usually, motile Aeromonas strains have not been susceptible to this chemotherapeutic (5, 12, 23, 27, 33). The distribution of resistance to cephalotin among the examined strains of motile Aeromonas was different in several species. A high percentage of the A . hydrophila (87% ) and A. caviae (83%) strains were resistant to this antibiotic, whereas the percentage of resistant A . sobria strains was lower (37%) . Motyl et al. (27) und Kuijper et al. (23) have also observed differences in susceptibility to cephalotin among motile Aeromonas strains . Motyl et al. (27) have suggested to use cephalotin susceptibility as a marker for A . sobria . Analysis of resistance results for each strain showed that the most common resistance patterns were for A. sobria : AMP, CAR (22% of strains ), AMP, CAR, SXT (24%) and AMP, CAR, CTN, CED (24%); for A . hydrophila: AMP, CAR, CTN, CED (48%) and AMP, CAR, CTN, CED, SXT (19%); for A . caviae: AMP, CAR, CTN, CED (35%) and AMP, CAR, CTN, CED, SXT (24% ). All of the strains being resistant to antibacterial agents were tested for transfer of their resistance to the recipients . Three strains transferred resistance to the A. hydrophila recipient . The transferred plasmids showed a stability ranging berween

ampicillin chloramphenicol colistin gentamicin kanamycin stre ptomycin tetracycline nalidixic acid

ampicillin chloramphenicol colistin gentamicin kanamycin streptomycin tetracycline nalidixic acid

ampicillin chloramphenicol colistin gentamicin kanamycin streptomycin tetracycline na lidixic acid

Aeromonas hydrophila (31)*

Aeromonas caviae (29)

Aeromonas sobria (46)

* N o of strains.

Antimicrobial agent

Species

98 74 91 9 83 100

91 11 11

76 98

69 97

97 97 97 86 7 83 100

97

94 100

55 69 48 28

97 42 94 7

1,56

87 7 48

:s;:0,78

98 91 98 41 2 83

97 100 97 100 48 97

97 94 100 29 7 97

3,13

65 77 97

2 98 98 100 85 57 83

79 97

100

97

3 97 94

6,25

97 100

100 97 97

93 100 2 100 98 98 100

90 97 2 98 98 100 98 85

100

94

94

98

98 100

4

100

7

97

97

3

100

2

97

97

3

3

50

3 100 94

MIC (ug/ml) 12,5 25

98

7

7

100

97

3

200

Table 1. Cumulative percentage of Aeromonas spp . inhibi ted by 8 antibacterial agents as determined by agar dilution method

100

100

100

100

100

~400

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0-

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0-

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::;;

::l 0

N

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00 00

susceptible inte rmediate resistant

suscep tible intermediate resistant

suscepti ble intermediate resistant

A . hydrophila (31)

A . caviae (29)

A. sobria (46 )

0 1 45

0 1 28

1* 1 29

CAR

42 0 4

28 0 1

31 0 0

AZL

45 1 0

29 0 0

31 0 0

46 0 0

29 0 0

31 0 0

Antibacterial agents : TOB AMK

46 0 0

29 0 0

31 0 0

NET

27 0 19

11

18 0

24 0 7

SXT

29 0 17

5 0 24

4 0 27

CTN

21 4 21

2 2 25

6 1 24

CED

46 0 0

29 0 0

31 0 0

CTX *'}

Symbols/abbreviations: CAR - carbenicillin, AZL - azlocillin, TOB - tobramycin, AMK - amikacin, NET - netilmicin, SXT - trimethopr imsulfamethoxazole, CTN - cephalotin, CED - cefradine, CTX - cefotaxime * No of strains. * * Identical results were noted for cefuroxime, ceftriaxone and cefoperazone. One strain of A. caviae and one str ain of A . sobria were resistant to cefamandole.

Degree of suscep tibi lity

Species (N o. of strains)

Table 2. Susceptibility of 106 strains of motile Aeromonas determined by the disk method

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AMP, CAR, CED *, CTN, COL, TET

AMP, CAR, CTN, TET, STR

AMP, CAR, CTN, CED, CMP, GEN, TET, STR, SXT

A. hydrophila AK 318 (lake water)

A . hydrophila AK 88 (lake water)

A. caviae AK 280 (sewage) 100; 1,5

100; 7,5; 4,8; 3,2 ; 3,0; 2,4; 2,0 ; 1,7 ; 1,5

60

Plasmid size (Md)

CMP, GEN, STR

TET, STR

TET

100

100

60

A . hydrophila Resistance Plasmid

TET

100*

E. coli Resistance Plasmid

Recipient

Symbols/abbreviations: AMP - ampicillin, CAR - carbenicillin, CED - cefradine, CTN - cephalotin, COL - colistin, TET - tetracycline, STR streptomycin, CMP - chloramphenicol, GEN - gentamicin, SXT - trimethoprim-sulfamethoxazole * Plasmid unstable in E . coli.

Resistance pattern

Strain (source)

Donor

Table 3. Transmissible R plasmids of motile Aeromonas strains

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0-

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Susceptibilities of Motile Aeromonas sp. to Antimicrobial Agents

91

98% and 100% . In only one case, resistance (to tetracycline) determined by the 100 Md plasmid, was transferred to the E. coli, but the plasmid was unstable in this recipient. More than 90% of the cells were plasmid-negative after only one subculture in drug-free medium. This plasmid could be maintained in the E. coli recipient grown in selective medium . Identical results were noted at 30 °C and at Transconjugants as well as donor strains were lysed and the molecular weights of the plasmids have been determined. The results are shown in Table 3. The plasmids which are transferred from A . hydrophila to E. coli are grouped into three classes; plasmids of incompatibility groups C, plasmids of incompatibility group U, and plasmids unstable in E. coli not belonging to any defined incompatibility group (15). In our study, we have found one plasmid which belonged to the third class and two plasmid being non transmissible to the E. coli recipient. The results indicate that some of the tested strains of Aeromonas sp. were resistant to antimicrobial agents and thus forming a potential reservoir of R plasmids in the environment. The transmission of resistance to antimicrobial agents represents a potential public health hazard. Our investigation of the plasmid transfer has shown that some plasmids of motile Aeromonas strains are transferable only between strains of the same species or genera .

3rc.

Acknowledgements. We are grateful to Prof. ]. De Ley, Prof. T. Lachowicz, Dr. M. Bobrowski and Dr. F. 1. Macrina for providing reference bacterial strains.

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Dr. Adam Kaznowski, Department of Microbiology, A. Mickiewicz Universiry, Fredry 10,61-701 Poznan, Poland