Comparative in vitro activities of 13 antimicrobial agents against Morganella-Proteus-Providencia group bacteria from urinary tract infections

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1987, 0066-4804/87/101644-04$02.00/0 Copyright C) 1987, American Society for Microbiology

p. 1644-1647

Vol. 31, No. 10

Comparative In Vitro Activities of 13 Antimicrobial Agents against Morganella-Proteus-Providencia Group Bacteria from Urinary Tract Infections RAFFAELE PICCOLOMINI,* LUIGINA CELLINI, NERINO ALLOCATI, ARTURO DI GIROLAMO, AND GIAMPIETRO RAVAGNAN Cattedra di Microbiologia, Istituto di Medicina Sperimentale, Facolta di Medicina e Chirurgia, Universitdi degli

Studi "G. D'Annunzio," I-66100 Chieti,

Italy

Received 26 June 1987/Accepted 7 July 1987

We examined 741 urinary tract isolates of the Morganella-Proteus-Providencia group, including the recently defined species Proteus penneri, for susceptibilities to aminoglycosides, semisynthetic penicillins, and cephalosporins. The data emphasize the importance of identification to the species level on the basis of marked species differences in patterns of susceptibility.

During the last few years, an increase in the frequency of antibiotic resistance among the Proteeae has been noted (4, 11). Therefore, continued concern about antimicrobial resistance is warranted, as are studies on the activities of new agents. Unfortunately, in studies of in vitro susceptibility, species of Proteeae are often divided into Proteus mirabilis, indole-positive Proteus spp., and Providencia spp. It has been shown that such broad groupings conceal major differences among species in susceptibilities to antimicrobial agents (11). For these reasons, we decided to perform a study of the in vitro susceptibilities of recently and precisely identified isolates of the Morganella-Proteus-Providencia group, including the recently defined species Proteus penneri (3, 5, 6, 9), by standardized susceptibility testing methods performed in our laboratory. A comparison was made between the in vitro activities of amikacin, tobramycin, carbenicillin, azlocillin, piperacillin, temocillin, cefazolin, cefoxitin, and cefuroxime and four newly developed preparations: aztreonam, imipenem, ceftazidime, and ceftriaxone. Laboratory-grade, antibiotic-standard powders were obtained from the manufacturers as follows: azlocillin, Bayer AG, Leverkusen-Bayer Italia, Milan, Italy; ampicillin, carbenicillin, and temocillin, Beecham Research Laboratories, Brentford, England; amikacin, Bristol Italiana SpA, Latina, Italy; piperacillin, Cyanamid Italia SpA, Catania, Italy; cefazolin and tobramycin, Eli Lilly Italia SpA, Sesto Fiorentino, Florence, Italy; cefuroxime and ceftazidime, Glaxo SpA, Verona, Italy; ceftriaxone, Roche SpA, Milan, Italy; and aztreonam, Squibb SpA, Rome, Italy. Stock solutions of antibiotics were prepared fresh and according to the instructions of the manufacturers. We examined the susceptibilities of 741 ampicillinresistant (MICs, .200 ,uglml) urinary tract isolates belonging to the Morganella-Proteus-Providencia group (Table 1) that had recently been isolated in our laboratory or sent to us from different areas of the Abruzzo region.

All of the organisms were isolated from urine specimens obtained from hospitalized patients with acute exacerbation of chronic urinary tract infection without urine flow obstruction but with underlying chronic pyelonephritis. Only one isolate from a patient was tested to avoid multiple copies of the same strain. Upon isolation or receipt, clinical isolates of Proteeae were fully identified and differentiated by standard procedures (2, 3, 5, 7, 8). All the strains were stored at -70°C, and fresh subcultures were used for susceptibility testing. MICs were determined for each antimicrobial agent in liquid medium by using a Titertek Motorized Multidiluter and Automatic Inoculator (Flow Laboratories SpA, Milan, Italy). Serial twofold dilutions were prepared at concentrations ranging from 200 to 0.025 jig/ml. The microdilution wells were filled with 100-,ul samples of the antibiotic solution prepared in Mueller-Hinton broth (BBL Microbiology Systems, Cockeysville, Md.) except for aminoglycosides, for which the medium was supplemented with calcium and magnesium ions (10). The volume of the inoculum was 100 ,ul, with a final bacterial concentration of 1.5 x 105 CFU/ml. Control strains, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, were simultaneously tested to ensure the potency of each drug. The microdilution plates were incubated overnight at 37°C. The trays were examined on a Titertek Plate Reading Box (Flow Laboratories). MICs were recorded as the lowest concentrations of antibiotic inhibiting visible growth. MIC ranges and MICs for 50 and 90% of the strains are shown in Table 1. At a concentration of 0.78 ,ug/ml, tobramycin inhibited, respectively, 50, 68, and 26% of the P. penneri, Providencia rettgeri, and Providencia stuartii strains. Providencia alcalifaciens and Proteus vulgaris were the most susceptible organisms.

P. vulgaris, P. rettgeri, and P. alcalifaciens, in that order, the most susceptible to amikacin. P. mirabilis showed susceptibility similar to that of P. stuartii, and P. penneri, for which MICs were similar to MICs for Morganella morganii, was the least susceptible.

were

*

Corresponding author. 1644

TABLE 1. Susceptibility patterns of tested Morganella-ProteusProvidencia group bacteria Organism (no. of isolates)

MIC

Antibiotic

(p.g/ml)'

Range

50%

90%

Morganella morgani (108)

Amikacin Tobramycin Cefoxitin Ceftazidime Ceftriaxone Cefuroxime Azlocillin Aztreonam Carbenicillin Imipenem Piperacillin Temocillin

1.56-50 0.2-0.78 1.56-25 0.05-3.12 0.025-0.78 0.2-12.5 25->200 0.39-12.5 1.56-100 0.1-6.25 1.56-100 50->200

3.12 0.39 6.25 0.1 0.025 1.56 50 0.78 3.12 1.56 12.5 100

25 0.78 12.5 1.56 0.2 12.5 100 6.25 50 6.25 100 200

Proteus

Amikacin Tobramycin Cefoxitin Ceftazidime Ceftriaxone Cefuroxime Azlocillin Aztreonam Carbenicillin Imipenem Piperacillin Temocillin

0.78-6.25 0.39-1.56 1.56-100 0.1-1.56 0.025-0.2 0.78-100 0.78->200 0.05-0.78 0.2->200 0.1-3.12 0.2->200 0.2->200

0.78 0.39 3.12 0.2 0.025 1.56 1.56 0.1 0.39 0.2 0.39 0.39

3.12 0.78 6.25 1.56 0.2 3.12 3.12 0.39 3.12 0.78 3.12 3.12

mirabilis (203)b

Organism (no. of isolates)

(39)c

Proteus

vulgaris (95)

Providencia

Amikacin Tobramycin Cefoxitin Ceftazidime Ceftriaxone Cefuroxime Azlocillin Aztreonam Carbenicillin Imipenem Piperacillin Temocillin Amikacin Tobramycin Cefoxitin Ceftazidime Ceftriaxone Cefuroxime Azlocillin Aztreonam Carbenicillin Imipenem Piperacillin Temocillin Amikacin

1.56-50

0.39-6.25 0.39-100 0.025-200 0.05-200 1.56->200 50->200 0.2-12.5 3.12-100 0.1-6.25 25-200 50->200

0.2-0.78 0.2-0.78 1.56-25

0.1-0.39 0.025-0.39 0.39-12.5 3.12-50 0.2-3.12 0.78-50 0.2-3.12 0.2-3.12 0.39-50 0.39-1.56

0.1-0.39 alcalifaciens Tobramycin 0.2-12.5 Cefoxitin (61) Ceftazidime 0.025-0.1 Ceftriaxone 200 0.2-6.25 0.78->200 0.39-12.5 0.39-50 0.78->200

1.56 0.78 1.56 0.1 0.2 6.25 100 0.78 6.25 0.39 50 100

25

3.12 12.5 0.78 1.56 100

100 6.25

10312 200 200

0.2 0.2 6.25 0.1 0.05 3.12 6.25 0.39 1.56 0.39 0.78 0.78

0.39 0.39 12.5 0.2 0.2 12.5

0.78 0.2 1.56 0.05 200 Cefoxitin 0.025-1.56 Ceftazidime Ceftriaxone 200 Carbenicillin 0.78-50 Imipenem 0.39-12.5 Piperacillin 0.39-100 Temocillin

Providencia stuartii (88)

Amikacin Tobramycin Cefoxitin Ceftazidime Ceftriaxone Cefuroxime Azlocillin Aztreonam Carbenicillin Imipenem Piperacillin Temocillin

a

Proteus penneri

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NOTES

VOL . 31, 1987

0.39-6.25 1.56-25 1.56-100 0.025-1.56 0.025-3.12 0.78-25 50->200 0.39-6.25 0.78->200 0.2-3.12 0.78-50 1.56->200

90% 1.56 3.12 50 0.2 0.2 12.5 12.5 6.25 3.12 12.5 3.12 3.12

0.39 0.39 3.12 0.05 200 3.12 50 3.12 12.5 50

50% and 90%, MIC for 50 and 90% of isolates, respectively. IIncluding 28 f3-lactamase producers (13.8%). Including one isolate resistant to all cephalosporins (MIC, >100

Ftg/ml).

See the text.

Substantial differences with respect to cephalosporin susceptibilities of various species were noted. P. mirabilis (including 28 P-lactamases producers) and P. alcalifaciens were differentiated from the other species by their susceptibilities to all the cephalosporins. Cefazolin, (data not shown), the least active cephalosporin against other species (MICs, -200 ,ug/ml), at 25 ,ug/ml inhibited more than 90% of these two species. M. morganii, P.

penneri, and P. vulgaris were similar in their susceptibilities to the cephalosporins tested, but the resistance of P. penneri to cefuroxime and the marked inhibitory activity of cefoxitin against this species distinguished P. penneri from the other Proteeae. Ceftazidime and ceftriaxone were clearly the most effective antibiotics against the strains tested, except P. penneri and P. stuartii isolates. P. stuartii generally was markedly more resistant to azlocillin than to carbenicillin, piperacillin, or temocillin, but some strains that were highly resistant to carbenicillin and

temocillin were susceptible to piperacillin. A similar differential susceptibility pattern was seen with P. alcalifaciens, but P. stuartii was much more resistant to both azlocillin and piperacillin than was P. alcalifaciens. The newly tested synthetic P-lactam antimicrobial agents aztreonam and imipenem had good activity against these species, with overall MICs for 90% of strains of 3.12 ,ug/ml. P. mirabilis was highly susceptible to all drugs (MICs for 90% of isolates, 0.39 to 3.12 jig/ml), with the exception of 13.8% of the isolates, which produced ,B-lactamases. These isolates were resistant to 200 ,ug of antibiotic per ml. P. vulgaris and P. rettgeri showed susceptibility patterns similar to that of P. mirabilis, with the exception of slightly increased resistance to azlocillin, aztreonam, carbenicillin,

1646

NOTES

and temocillin (P. vulgaris) and azlocillin, aztreonam, and imipenem (P. rettgeri). Although only 39 P. penneri isolates were available for this study, they seemed to be markedly more resistant to all the penicillins tested than was P. vulgaris. M. morganii strains had a susceptibility pattern very similar to that of the P. penneri strains. Among the Proteeae studied, a remarkable difference in activity was noted for one P. penneri strain, which required up to 100 ,ug of ceftazidime or ceftriaxone per ml to be inhibited but was susceptible to 0.78 ,ug of imipenem per ml. This resistant P. penneri strain was obtained from a patient who had been treated with ceftriaxone. This was a woman with chronic interstitial nephritis and vesicoureteral reflux from whom a ceftriaxone-susceptible strain of P. penneri (MIC, 0.78 ,ug/ml) was isolated in pure culture (>105 CFU/ml) from a urine specimen before antimicrobial treatment. The patient received ceftriaxone at an intramuscular dose of 1 g every 24 h for 7 days and showed a prompt clinical response, but at 10 days posttherapy she relapsed with a recurrence of her urinary tract infection. From a urine specimen obtained at this time, a pure growth of >105 CFU of P. penneri per ml was again obtained, and this strain was found to be resistant to ceftriaxone and the other cephalosporins, with MICs increasing eightfold for ceftriaxone and ceftazidime, over fivefold for cefuroxime, and twofold for cefoxitin. Serotyping conducted in our laboratory strongly suggested that the susceptible and resistant isolates were the same strain. Major differences in susceptibilities to aminoglycosides, carbenicillinlike ,3-lactam antibiotics, and cephalosporins among Morganella-Proteus-Providencia group bacteria were demonstrated in this study. P. alcalifaciens and P. mirabilis were readily distinguishable by their marked susceptibilities to aminoglycosides and cephalosporins. P. stuartii was found to be typically resistant to tobramycin, whereas P. alcalifaciens was susceptible; furthermore, our data proved that Providencia spp. are susceptible to amikacin. In addition, marked differences between the susceptibilities of these three species to cephalosporins are reported here, and we found that these differences extend to all the modified penicillins that were tested in this study. Interestingly, P. penneri was more resistant to the modified penicillins than was P. vulgaris. This behavior was consistent with previously reported antimicrobial susceptibilities to carbenicillin (4, 5). Moreover, the higher resistance of P. penneri to cefuroxime clearly separated it from the other six species. It is noteworthy that among the 39 P. penneri strains isolated in the Abruzzo region, one strain, derived from a patient who failed clinically during treatment with ceftriaxone, was resistant to broad-spectrum cephalosporins. The emergence of such mutants, reported in vitro and in vivo by several researchers, especially in Enterobacter and Pseudomonas spp. (1, 13, 16), was most probably due to the derepression of the inducible cephalosporinase during constitutive synthesis (14). Overproduced P-lactamase acts by hydrolyzing the cephalosporins or by merely trapping them, thereby creating an enzymatic impenetrability of the cephalosporins into the periplasmic space of bacterial cells (15). In the present study, imipenem proved to be the most potent compound against P. penneri strains with derepressed 13-lactamase and thus seemed to be unaffected by the derepression of chromosomal P-lactamases, a statement also made by Sanders (12).

ANTIMICROB. AGENTS CHEMOTHER.

With regard again to P. penneri, it is remarkable that this heretofore unknown species, isolated with some frequency in various laboratories, must be considered, although it is difficult to accept that it would not have been detected as an atypical indole-negative P. vulgaris strain or an atypical ornithine decarboxylase-negative P. mirabilis strain. It is likely that it has been isolated just as frequently before and was overlooked or misidentified. In conclusion, the results of this in vitro study authorize us to affirm, in agreement with Hawkey et al. (4), that accurate identification to the species level of isolates of Proteeae in surveys of susceptibilities to antimicrobial agents is essential if potentially misleading results are not to be reported. This work was supported by grant 86.01619.52 from the Consiglio Nazionale delle Ricerche (Italy). We thank Barbara Buzzelli and Angela Del Vecchio of the microbiology laboratories of Chieti and Pescara General Hospitals for supplying some of the bacterial isolates and Giovanni Catamo for his technical assistance. LITERATURE CITED 1. Coliatz, E., L. Gutmann, R. Williamson, and J. F. Acar. 1984. Development of resistance to P-lactam antibiotics with special reference to third-generation cephalosporins. J. Antimicrob.

Chemother. 14(Suppl. B):13-21. 2. Cowan, S. T. 1974. Cowan and Steel's manual for the identification of medical bacteria, 2nd ed. Cambridge University Press,

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8. Michael, T. K., D. J. Brenner, and J. J. Farmer III. 1985. Enterobacteriaceae, p. 263-277. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 9. Muller, H. E. 1986. Occurrence and pathogenic role of Morganella-Proteus-Providencia group bacteria in human feces. J. Clin. Microbiol. 23:404-405. 10. National Committee for Clinical Laboratory Standards. 1983. Tentative standard M7-T. Standard methods for dilution antimicrobial susceptibility tests for bacteria which grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 11. Penner, J. L., M. A. Preston, J. N. Hennessy, L. J. Barton, and M. M. Goodbody. 1982. Species differences in susceptibilities of Proteeae spp. to six cephalosporins and three aminoglycosides. Antimicrob. Agents Chemother. 22:218-221. 12. Sanders, C. C. 1984. Inducible P-lactamases and non-hydrolitic resistance mechanisms. J. Antimicrob. Chemother. 13:1-3.

VOL. 31, 1987 13. Sanders, C. C., and W. E. Sanders, Jr. 1983. Emergence of resistance during therapy with the newer 1-lactam antibiotics: role of inducible ,-lactamases and implications for the future. Rev. Infect. Dis. 5:639-648. 14. Seeberg, A. H., R. M. Tolxdorff-Neutzling, and B. Wiedemann. 1983. Chromosomal 1-lactamases of Enterobacter cloacae are responsible for resistance to third-generation cephalosporins.

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Antimicrob. Agents Chemother. 23:918-925. 15. Then, R. L., and P. Angehrn. 1982. Trapping of nonhydrolyzable cephalosporins by cephalosporinases in Enterobacter cloacae and Pseudomonas aeruginosa 'as a possible resistance mechanism. Antimicrob. Agents Chemother. 21:711-717. 16. Towner, K. J. 1984. Emerging bacterial resistance to the newer cephalosporins. J. Antimicrob. Chemother. 13:526-527.