BARTLEY, NANCY C. JOYCE, AND VINCENT T. ANDRIOLE*. Departments of Medicine and Pathology, Hartford Hospital, Hartford, Connecticut 06155; ...
Vol. 8, No. 1 Printed in U.S.A.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1975, p. 54-57 Copyright 0 1975 American Society for Microbiology
Activity of Minocycline Against Acinetobacter calcoaceticus Var. anitratus (Syn. Herellea vaginicola) and Serratia Marcescens EUFRONIO G. MADERAZO, RICHARD QUINTILIANI, RICHARD C. TILTON, RPAYMOND BARTLEY, NANCY C. JOYCE, AND VINCENT T. ANDRIOLE* Departments of Medicine and Pathology, Hartford Hospital, Hartford, Connecticut 06155; Department of Laboratory Medicine, University of Connecticut Health Center, Farmington, Connecticut 06032; and Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510* Received for publication 10 March 1975
The activity of minocycline and tetracycline against 23 isolates of Acinetobacter calcoaceticus var. anitratus (syn. Herellea vaginicola) and 178 strains of Serratia marcescens was determined by disk and microdilution methods. The results indicate that minocycline is highly active against this species of Acinetobacter, all but one strain being inhibited by 0.007 jig of the antibiotic per ml. Tetracycline was also active, though to a lesser degree, against A. calcoaceticus. Of the 178 strains of S. marcescens tested, only seven (3.9%) had a minimum inhibitory concentration of 2 gg or less of minocycline per ml. Tetracycline was less active than minocycline against S. marcescens; with 2 ,ug of tetracycline per ml, only 2 of 152 (1.3%) strains were inhibited. At concentrations of 8 and 16 Mg of minocycline per ml, which can be achieved in the urine with the usual doses, 44.9 and 63.5% of S. marcescens strains were inhibited, which implies its possible usefulness for the therapy of urinary tract infection due to
this organism. Observations implicating Acinetobacter calcoaceticus var., anitratus (syn. Herellea vaginicola) and Serratia marcescens in clinical infections have recently been noted more frequently (1, 4, 5, 6, 8, 10, 13, 22). (Acinetobacter calcoaceticus var. anitratus will be referred to subsequently as A. calcoaceticus). Of much concern in these infections is the associated high mortality. This is most likely due to a combination of many factors, among which are the organisms' virulence when infecting debilitated hospitalized patients and their resistance to most antibiotics. Both organisms are often susceptible only to the relatively more toxic antibiotics such as kanamycin and gentamicin. Since the introduction of minocycline (7-dimethyl-amino-6-demethyl-6-deoxytetracycline hydrochloride), a new semisynthetic tetracycline, we have encountered A. calcoaceticus and S. marcescens strains susceptible to this antibiotic. Accordingly, a more thorough investigation of the in vitro susceptibility of these organisms to minocycline was undertaken.
criteria of Tatum et al. (19). The 178 S. marcescens isolates tested were identified by the method of Edwards and Ewing (7), and 120 were isolated from patients admitted to the Yale-New Haven Hospital, New Haven, Conn. The remainder were obtained from various hospitals in Connecticut. Disk susceptibility testing. The method used was that described by Bauer et al. (3) and is detailed in the Federal Register (9). Minocycline disks were obtained from Lederle Laboratories (Pearl River, N.Y.) and contained 30 Mg of antibiotic per disk. The 30-,g tetracycline disks were purchased from Baltimore Biological Laboratories (Baltimore, Md.). All organisms were tested against minocycline. One hundred and fifty strains of S. marcescens were additionally tested against tetracycline disks, as were 21 strains ofA. calcoaceticus. Minimal inhibitory concentration. The minimal inhibitory concentration (MIC) was determined by using a previously described microdilution technique (2, 20). Freshly prepared Mueller-Hinton broth medium (MH) was used in all experiments. In 152 of 178
strains of S. marcescens tested, additional experi-
ments were done by using brain heart infusion broth (BHI) as the culture medium. Organisms were considered to be susceptible if their growth was inhibited by 2 gg/ml or less since this concentration can be MATERIALS AND METHODS achieved in the serum after administration of a single Bacterial strains. The 23 isolates of A. standard oral dose of these antibiotics. The data were calcoaceticus used in this study were obtained from also analyzed in relation to concentrations of these clinical specimens submitted to two hospitals in antibiotics which are attainable in the urine. In this Hartford, Conn., and were identified according to the context, organisms were considered susceptible if 54
their growth was inhibited by concentrations of 16 Statistics. MIC values in micrograms per milliliter were expressed as the log2 MIC. The interconversion units in expressing the dilution interval as micrograms per millliter and log2 micrograms per milliliter are shown in Table 1. The Wilcoxon matched-pairs signed-rank test was employed to determine probability values (16).
RESULTS A. calcoaceticus. Disk diffusion testing of A. calcoaceticus showed inhibition zones of 23 to 30 mm with minocycline and 16 to 29 mm with tetracycline. The MIC of minocycline for 22 of the 23 strains tested was less than 0.007 gg/ml. One strain was inhibited by a concentration of 0.5 ,ug of the antibiotic per ml. Tube dilutions of tetracycline were tested for 21 strains; the MIC was 0.125 ug/ml for 9, 0.25 Ag/ml for 7, and 0.5 ,ug/ml for 5 strains. All 23 isolates of A. calcoaceticus were exquisitely susceptible to minocycline, and all 21 strains tested were also susceptible, though to a slightly lesser degree, to tetracycline. S. marcescens. Fig. 1 (A and B) compares the zones of inhibition by disk susceptibility testing with the MICs of tetracycline (152 strains) and minocycline (178 strains) for S. marcescens. Only 7 of 178 (3.9%) and 2 of 152 (1.3%) strains of S. marcescens were susceptible to 2 ug/ml or less of minocycline and tetracycline, respectively. In the present study, the comparison of MICs of minocycline against the diameter of inhibition observed by disk susceptibility testing suggests that a zone of inhibition of 19 mm or more is required to define strains of S. marcescens as susceptible to minocycline by disk testing. The present observations indicate that four of seven strains, shown to be susceptible to minocycline by the MIC technique, could be correctly identified and, more importantly, no resistant strains were misTABLE 1. Interconversion units for expressing MIC as dilution interval in ug/ml and log2,g/ml
1 2 3 4 5 6 7 8 9
Antibiotic concn
,ug/ml
log2 zg/ml
>128 128 64 32 16 8 4
>7
2 1
7 6 5 4 3 2 1 0
MWKLLES-NOTON SlOTH
1n28
jig/ml or less (17).
Dilution interval
55
MINOCYCLINE AGAINST HERELLEA AND SERRATIA
VOL. 8, 1975
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B ZONE Of INHIBITION (_) FIG. 1. Disk susceptibility testing with MICs of tetracycline and minocycline for S. marcescens. identified as susceptible (Fig. 1A). With tetracycline (Fig. 1B), neither of the two susceptible strains was correctly identified as such by disk testing. A susceptible zone of inhibition for tetracycline 30-,gg disks against S. marcescens could not be accurately defined in the present studies. These observations suggest that it might be appropriate for clinical laboratories to include minocycline disks, in addition to tetracycline, when determining antibiotic sensitivities to S. marcescens. Table 2 lists the MICs of tetracycline and minocycline for S. marcescens tested in two different culture media. If antibiotic levels easily achievable in the urine, such as 8 ,ug/ml, were used as the MIC breakpoint for susceptibility, 80 of 178 (44.9%) strains of
56
ANTIMICROB. AGENTS CHEMOTHER.
MADERAZO ET AL.
TABLE 3. Differences of observed mean MIC of S. marcescens tested would be susceptible to tetracycline and minocycline by using different minocycline and 25 of 152 (16.5%) to tetragrowth media cycline; at 16 ,g of minocycline per ml, 113 of 178 (63.5%) strains were inhibited. With either Mean MIC (log2 ug/ml) medium, the MIC of tetracycline is greater than Antibiotic BHI vS. that of minocycline. This difference of mean BHvs MH BHI MH MIC is significant (P < 0.001) for both BHI and MH (Table 3). If MICs of each antibiotic 0.44 5.28 5.72 were compared for differences obtained with Tetracycline (P < 0.001) the use of the two different media, the MICs Minocycline 0.57 3.94 4.51 were significantly higher (P < 0.001) when BHI (P 128 128 64 32 16 8 4 2 1
Tetracycline (N= 152) Cumula- Cumulative no. of strains inhibited
tive % of strains inhibited
152 138 81 63 35 25 10 2 0
100 90.8 53.3 41.5 23.1 16.5 6.6 1.3 0
may be minimized by lower doses (50 mg) of minocycline given at 6-h intervals (C. Hodgson, personal communication; V. T. Andriole, unpublished observation). Furthermore, although tetracycline is not as active as minocycline in this study, the tetracycline MICs for A. calcoaceticus are low and should be easily achieved in blood. These results are unlike those obtained by Steigbigel et al. (18) who found only one of seven strains inhibited by 6.3 gg of tetracycline per ml. In contrast to its effect on A. calcoaceticus, minocycline is not nearly as active against S. marcescens, only 7 of 178 strains (3.9%) having been inhibited by 2 Mg of the antibiotic per ml (15.7% by 4 ,g/ml and 44.9% by 8 ,ug/ml). These results resemble that reported by McGowan et al., who found 4 of 13 strains of S. marcescens to be inhibited by 6.3 to 12.5 Mg of minocycline per ml (12). Different results were obtained by Washington et al. (21), who found 95% of 45 isolates susceptible to 3.12 Mg of the antibiotic per ml. The reason for this discrepancy is
marcescens to tetracycline and minocycline by using MH and BHI broths BH1
Minocycline (N= 178) CumulaCumulative % of tive no. of strains strains inhibited inhibited
f78 176 156 113 80 28 7 2
100.0 98.9 87.7 63.5 44.9 15.7 3.9 1.1
Tetracycline (N = 151) CumulaCumulative % of tive no. strains of strains inhibited inhibited
151 111 76 62 31 14 3 1 0
100 73.5 50.3 41.0 20.5 9.2 1.9 0.6 0
Minocyline (N = 151) Cumula- Cumulative % of tive no. strains of strains inhibited inhibited
151 144 113 64 26 3 3 1
100 95.4 74.9 42.4 17.2 2.0 2.0 0.7
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MINOCYCLINE AGAINST HERELLEA AND SERRATIA
uncertain since the conditions used (medium and inoculum) in the two studies were similar. It is possible that this is an example of geographic variance between strains. These observations demand caution when minocycline therapy is contemplated for S. marcescens infections, especially when it occurs outside the urinary tract. Obviously, minocycline should be considered only for isolates that have been determined susceptible by either disk or tube dilution testing. With concentrations of 8 to 16 ,g of minocycline per ml, levels which could be achieved in the urine by the usual oral doses (18), 44.9 to 63.5% of S. marcescens strains tested were inhibited. Since the majority of S. marcescens infections occur in the urinary tract (14), this finding may have a useful clinical application. Differences in MIC values for various antibiotics which result from the use of different growth media have been known for both disk diffusion (11) and tube dilution tests (20). The findings of this study indicate a higher MIC for both tetracycline and minocycline when BHI instead of MH is used as the growth medium. This effect is greater for minocycline than for tetracycline, and may reflect the effect of differences in divalent cationic content (Mg2+ and Ca2+) of the two media (20, 21). ACKNOWLEDGMENT This study was supported by grant 73-851 from the American Heart Association. LITERATURE CITED 1. Altemeir, W. A., W. R. Culbertson, W. D. Fuller, and J. J. McDonough. 1969. Serratia marcescens septicemia. A new threat in surgery. Arch. Surg. 99:232-238. 2. Andriole, V. T. 1971. Synergy of carbenicillin and gentamicin in experimental infections with pseudomonas. J. Infect. Dis. 124(Suppl.):546-555. 3. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turek. 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45:493-496. 4. Bodey, G. P., V. Rodriguez, and J. P. Smith. 1970. Serratia sp. infections in cancer patients. Cancer 25:199-205. 5. Crowder, J. G., G. H. Gilkey, and A. C. White. 1971. Serratia marcescens bacteremia. Clinical observation
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