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Antonie van Leeuwenhoek 53:253-259 (1987) 9 Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Susceptibility to mercurials of clinical Pseudomonas aeruginosa isolated in M6xico CARLOS CERVANTES-VEGA 1& JAIME CHAVEZ Instituto de Investigaciones Qulmico-Biolrgicas, UniversidadMichoacana, 58240, Mordia, Michoacim, Mkxico; (lpresent address: Department of Microbiology and Immunology, University of Illinois College of Medicine, P.O. Box 6998, Chicago, 1L 60680 USA ) Received 05 January 1987; accepted 29 April 1987

Key words: P. aeruginosa, mercury, organomercurials, pyocin types, antibiotic resistance, metal resistance Abstract. Susceptibility to inorganic mercuric ions and to organomercurials of 237 Pseudomonas aeruginosa clinical strains isolated in Mexico was determined by agar dilution tests. Resistant strains fell into two classes: i) narrow-spectrum resistant strains (27~o of total isolates) resistant only to mercuric ions and to merbromin, and most grouped in pyocin type 1; and ii) broad-spectrum resistant strains (5~o) with additional resistances to thimerosal, phenylmercury, methylmercury and p-hydroxymercuribenzoate, that belonged mostly to pyocin type 10. Mercurial resistant isolates showed a higher proportion of resistance to antibiotics and metals than did mercurial sensitive isolates, and broad-spectrum resistant strains had the highest frequency of resistance to antibiotics and to tellurite and arsenate.

Introduction The emergence of antibiotic resistance in clinical bacteria has been explained by the immoderate use of antibiotics in the hospitals. The appearance of clinical isolates resistant to heavy metals (Novick & Roth 1968; Silver 1981; Porter et al. 1982) is, however, not completely understood. Antibiotic and metal resistances are often determined extrachromosomally and can be found associated in the same plasmid (Novick & Roth 1968; Groves & Young 1975; Nakahara et al. 1977a, b; Summers et al. 1978; Cenci et al. 1982). Resistance to mercury (Hg 2+) and organomercurials has been found in bacteria isolated from contaminated soil and water areas (Nelson et al. 1973; Walker & Colwell 1974; Summers et al. 1978; Timoney et al. 1978; Khesin & Karasyova 1984) but resistant clinical strains are also commonly encountered (Groves & Young 1975; Nakahara et al. 1977a, b; Cenci et al. 1982; Porter et al. 1982); bacterial mechanisms for mercurial detoxification have been established (Silver 1981; Robinson & Tuovinen 1984). Pseudomonas aeruginosa is an opportunistic pathogen whose antimicrobial

254 Carlos Cervantes- Vega & Jaime Chhvez resistance constitutes a major factor for its prevalence in hospital settings. We report here the susceptibility to mercurials of P. aeruginosa clinical isolates and relate these data with resistance to antibiotics and metals.

Materials and methods

Bacterial strains A group of 237 isolates ofP. aeruginosa was collected during the period of April, 1980-December, 1983 in Morelia, Michoacan, Mexico from infected patients at four hospitals and three private clinical laboratories. Isolates were mostly obtained from wounds, urinary tract infections and ear secretions; multiple isolates from the same individual were excluded. Identification was accomplished by the following tests: growth on cetrimide agar, pyocyanin production in Tech agar, growth at 42 ~ oxidase test and gelatin hydrolysis (Cowan 1974).

Indicator strains Indicator strains for pyocin typing were provided by T. L. Pitt, Central Public Health Laboratory, London, England.

Media and reagents Culture media were obtained from Bioxon de Mexico; inorganic salts were from Merck. Merbromin (Mercurochrome), thimerosal (Merthiolate), phenylmercuric acetate, and p-hydroxymercuribenzoatewere a gift of S. Silver, Washington University, St. Louis, MO, USA. Methylmercuric chloride was provided by L. Sosa., Universidad de Guanajuato, Mexico.

Pyocin typing The cross-streaking procedure, as described by Govan (1978) was used to type the clinical isolates.

Pseudomonas aeruginosa 255 Susceptibility testing Minimal inhibitory concentration (MIC) of mercurials and metals were determined by dilution tests in nutrient agar; for antibiotics, Mueller-Hinton agar was used. Inoculation of overnight broth cultures was according to Ericsson & Sherris (1971). MIC was defined as the lowest concentration of the compound inhibiting visible growth after an incubation of 24 h at 37 ~ Criteria for resistance was based on MIC frequency distribution in the case of metals and on achievable serum levels for antibiotics (Cervantes-Vega et al. 1986).

Results

Distribution of MICs of mercurials is shown in Fig. 1; the values for considering clinical strains as resistant were obtained from MIC distribution frequencies. Bimodal curves of HgCI2 and merbromin (Fig. 1A, 1B) allowed a clear distinction between sensitive and resistant strains. All isolates resistant to HgC12 were also merbromin-resistant, and all organomercurial-resistant isolates showed resistance to inorganic mercury. MIC distribution curves of the remaining organomercurials show a small group of resistant isolates (Figs. 1C through IF), although for p-hydroxymercuribenzoate (Phmb) sensitive and resistant isolates were hardly distinguished (Fig. IF; six strains clearly resistant had a MIC of 380 #g ml 1; five strains, with MIC of 190 pg m1-1 and resistant to the other mercurials, were taken as Phmb-resistant; and 27 strains, also with a MIC of 190/tg ml-I but sensitive to other organomercurials were considered as Phmbsensitive). The results of mercurial susceptibility testing show that resistant isolates fell into two classes: - narrow-spectrum resistant strains, resistant only to mercury and merbromin broad-spectrum resistant strains, resistant to all mercurials tested (Table 1) Pyocin type distribution of the clinical isolates of the different susceptibility classes is also shown in Table 1. Sixty percent of narrow-spectrum resistant strains belonged to pyocin-type 1 and 90~ of broad-spectrum resistant strains were grouped in pyocin types 10 and 1. Tables 2 shows the proportions of antibiotic and metal resistance of the P. aeruginosa clinical isolates. Mercurial-resistant isolates, particularly broad-spectrum resistant strains, showed a higher frequency of resistance than did mercurial-sensitive strains; moreover, most pyocin type 10 isolates of broad-spectrum resistant class showed multiple resistance to both antibiotics and metals (data not shown).

256 Carlos Cervantes-Vega & Jaime Chhvez 60

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MIC (jJglml) Fig. 1. Minimal inhibitory concentration (MIC) distribution curves of clinical P. aeruginosa isolates. A - HgC12; B - merbromin; C - thimerosal; D - phenyl mercuric acetate; E - methyl mercuric chloride; F - p-hydroxymercuribenzoate. Arrows indicate concentrations above which isolates were considered as resistant.

Discussion

Almost a third (32~) of the P. aeruginosa isolates tested showed resistance to inorganic mercury (Table 1). Frequencies of mercury resistance in clinical bacteria are commonly high (Hall 1970; Nakahara et al. 1977a, b; Witte et al. 1980; Cenci et al. 1982; Porter et al. 1982). Nakahara et al. (1977a), for example, found

Pseudomonas aeruginosa 257 Table 1. Distribution of pyocin types ofP. aeruginosa clinical isolates in relation to mercurial susceptibility. Susceptibility class

No. o f isolates belonging to pyocin type (~)

Sensitive Resistant Narrow-spectrum a Broad-spectrum b

1

3

10

5

Others c

Total

58 (36)

35 (22)

12 (7)

10 (6)

48 (29)

163 (69)

38 (60) 3 (27)

7 (11) 1 (9)

12 (19) 0

63 (27) 11 (5)

6 (10) 7 (64)

0 0

a Resistant to HgC 12 and merbromin. b Resistant to all mercurials tested. c Includes isolates of 17 additional pyocin types.

that 7570 of P. aeruginosa clinical strains isolated in Japan were mercury-resistant. As previously found in E. coli and P. aeruginosa strains (Silver 1981), we encountered two classes of resistant strains: those resistant only to mercuric ions and merbromin (designated as narrow-spectrum strains) and those having additional resistances to thimerosal, phenylmercury, methylmercury, and p-hydroxymercuribenzoate (or broad-spectrum resistant strains). Resistance to p-hydroxymercuribenzoate has been reported associated with narrow-spectrum resistant plasmids in P. aeruginosa (Weiss et al. 1978) but we observed no clear distinction between sensitive and resistant isolates (Fig. IF). Narrow-spectrum resistant strains comprised 2770 of total isolates and 6070 belonged to pyocin type 1 (Table 1), the most commonly found pyocin type (Govan 1978). Broad-spectrum resistance strains were found at lower proportion and most belonged to pyocin type 10 (Table l). We have found that type 10 isolates are also more frequently resistant to antibiotics than those of other types (Cervantes-Vega et al. 1986). Table 2. Antibiotic and metal resistance o f P. aeruginosa clinical isolates in relation to mercurial susceptibility. Susceptibility class

Sensitive Resistant Narrow-spectrum Broad-spectrum

No. of resistant isolates (~) Sm a

Gm

Tb

Cb

Ter

Asa

Asi

Chr

67 (41)

6 (4)

0

3 (2)

10 (6)

6 (4)

5 (3)

2 (1)

33 (52) 9 (82)

6 (10) 4 (36)

5 (8) 3 (27)

2 (3) 4 (36)

14 (22) 4 (36)

17 (27) 4 (36)

11 (17) 1 (9)

9 (14) 0

asm - streptomycin; G m - gentamycin; Tb - tobramycin; Cb - carbenicillin; Ter - potassium tellurite; Asa - sodium arsenate; Asi - sodium arsenite; Chr - potassium chromate.

258 Carlos Cervantes- Vega & Jaime Chhvez Mercury-resistant isolates were more frequently resistant to antibiotics and metals than mercury-sensitive ones (Table 2), as reported by others (Nakahara et al. 1977a, b; Summers et al. 1978; Witte et al. 1980; Cenci et al. 1982). Broadspectrum resistant strains, on the other hand, showed the highest frequency of resistance to antibiotics and to teUurite and arsenate (Table 2); most broadspectrum resistant strains ofpyocin type 10 showed multiple resistance to antibiotics and metals. Selection for mercurial resistance in clinical bacteria has been explained by three alternatives (Robinson & Tuovinen 1984): - selection by use of mercurials in the hospitals (Nakahara et al. 1977a; Porter et al. 1982) - selection by extrahospitalary mercurial pollution (Hall 1970; Summers et al. 1978; Khesin & Karasyova 1984) - indirect selection by association of mercurial resistance and antibiotic resistance on R plasmids (Hall 1970; Groves & Young 1975; Witte et al. 1980) We have no precise data on mercurial consumption in Mexico, but organomercurial disinfectants (notably thimerosal) are still widely used. Maybe a mixed mechanism functions in selecting resistance, i.e. mercurial pollution selects for environmental resistant bacteria, which are then carried to the hospitals. There, either mercurials select for resistant strains or mercurial resistance genes recombine with R plasmids which, in turn, are selected by antibiotics. In this way, both mercurials and antibiotics may exert selective pressure for mercurial resistance. Analysis of mercurial susceptibility of bacteria from different origins will help to understand the rise and spread of resistance to mercurials.

Acknowledgments We thank S. Silver for providing mercurials and for a critical reading of the manuscript, and L. Sosa and S. Vaca for helpful discussions. We also thank T. L. Pitt for supplying bacterial strains, and L. Piedra for typing the manuscript. This work was partially supported by grant PCSABNA-002181 from the Consejo Nacional de Ciencia y Tecnologia (M~xico).

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P s e u d o m o n a s aeruginosa 259

Cambridge University Press, Cambridge Ericsson, H. M. & J. C. Sherris (1971) Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathologica Microbiologica Scandinavica (Section B Supplement) 217: 1-90

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