Examination of Pseudomonas aeruginosa-Gentamicin by tpf49254


									ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1982, p. 412-415                                         Vol. 21, No. 3

     Examination of Pseudomonas aeruginosa-Gentamicin
   Discrepancies Encountered in an Autobac I-Disk Diffusion
  Diagnostic Microbiology Laboratory, Memorial Hospital, and Infectious Disease Service, Department of
              Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
                           Received 31 August 1981/Accepted 29 December 1981

            Seven Pseudomonas aeruginosa strains found to be susceptible to gentamicin
         by the Autobac I system and resistant by the Bauer-Kirby disk diffusion method
         were tested for the presence of mixed populations of cells. Double zones of
         inhibition randomly appeared on gentamicin disk diffusion plates, and susceptible
         and resistant subpopulations were isolated from these plates. Growth kinetic

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         studies of separated strains and mixed subpopulations indicated that the suscepti-
         ble organisms grew rapidly and were read as susceptible at 5 h with the Autobac I
         system. Resistant organisms entered a sustained lag phase and did not achieve
         sufficient turbidity to be read as resistant with the Autobac I system before 6 h.
         Thus, a false reading of susceptible could be obtained with the Autobac I system,
         depending on the ratio of susceptible organisms to resistant organisms selected for
         testing. A mixed susceptible and resistant population could be recognized by
         extending the incubation time of the Autobac I cuvette or by using other
         susceptibility methods to test isolates with light-scattering indexes of <1.0.

   The Autobac I system (General Diagnostics)          difficulty identifying methicillin-resistant staph-
is a semiautomated system for rapid (3 to 5 h),        ylococci and gentamicin-resistant P. aeruginosa
semiquantitative susceptibility testing of bacte-      strains (7). These organisms were rarely seen in
ria. In an initial collaborative study, the per-       this country at that time. Subsequent reports
formance of this system was shown to have a            have shown that the Autobac I system indeed
high overall level of agreement (91.5%) with the       had difficulty detecting methicillin-resistant
disk diffusion method (7). However, a number of        Staphylococcus aureus (1, 3). In one study (1),
discrepancies have been reported for several           the authors were able to demonstrate that there
organism-antibiotic combinations (1, 3, 4, 6, 7).      were mixed populations of susceptible and resis-
   In a preliminary study in our laboratory (T. E.     tant organisms within their S. aureus cultures.
Kiehn, J. B. Mayo, and D. Armstrong, Abstr.            When we began to study the Pseudomonas
Annu. Meet. Am. Soc. Microbiol. 1979, C75, p.          isolates that gave discrepant susceptibility test
322), the antibiotic susceptibilities of 1,033         results, the following two observations suggest-
gram-negative clinical isolates were determined        ed that we might also be working with mixed
by both Autobac I and Bauer-Kirby disk diffu-          populations: (i) double zones of growth were
sion methods. We encountered 42 discrepancies          found around some of the gentamicin disks on
which were confirmed by repeated testing. The          the disk diffusion plates, and (ii) the results of
most prevalent discrepancy was observed when           the tests often changed back and forth between
we tested the susceptibility of Pseudomonas            susceptible and resistant on repetitive testing. In
aeruginosa isolates to gentamicin; the Autobac I       this paper we report the results of studies under-
system indicated that the organisms were sus-          taken to explain the discrepancies which we
ceptible, whereas the disk diffusion and broth         observed when we performed tests on the sus-
dilution methods indicated that the organisms          ceptibility of P. aeruginosa to gentamicin.
were resistant. These inconsistencies when the
Autobac I system was used were particularly                            MATERIALS AND METHODS
disturbing because gentamicin is used common-                 Organisms. Suspensions of the seven P. aeruginosa-
ly to treat Pseudomonas infections in our hospi-           gentamicin discrepant isolates were preserved for fur-
tal.                                                       ther study in sterile, defibrinated sheep blood at -70°C
   The authors of the original collaborative study         until further testing. The organisms were thawed and
suggested that the Autobac I system might have             subcultured on Columbia agar plates containing 5%
VOL. 21, 1982                                    AUTOBAC I-DISK DIFFUSION DISCREPANCIES                    413
sheep blood. The plates were incubated overnight at        isms were determined by comparing the number of
35°C. The control strains used throughout the study        colonies on plates containing gentamicin with the
were Escherichia coli ATCC 25922 and ATCC 29194            number of colonies on the antibiotic-free plates.
and P. aeruginosa ATCC 27853.
   Susceptibility test methods. The Autobac I system                           RESULTS
was used according to the instructions of the manufac-
turer, except where indicated. The susceptibility pro-        A series of gentamicin disk diffusion plates
cedures outlined in the original collaborative evalua-     were inoculated with each discrepant Pseudo-
tion of the Autobac I system were followed (7). The        monas isolate in an attempt to demonstrate
Autobac I photometer automatically measured the            mixed susceptible and resistant populations.
extent of inhibition of growth by the antimicrobial        Each plate exhibited a susceptible zone of inhi-
agent compared with the growth control. This ratio
was expressed as a numerical value between 0 and 1,        bition, no zone of inhibition, or double zones of
called the light-scattering index (LSI). An LSI of .0.6    inhibition. The mixed nature of the isolates
was used to define susceptibility, and an LSI of <0.5      showing double zones of inhibition was con-
was used to define resistance. Values ranging from         firmed by Autobac I, disk diffusion, and broth
0.51 to 0.59 were defined as showing intermediate          dilution susceptibility test methods. The genta-
susceptibility.                                            micin-susceptible subpopulations gave LSI
   The standardized agar disk diffusion method was         readings of 0.68 to 1.0, disk diffusion zone sizes
performed as recommended by the National Commit-           of 15 to 17 mm, and minimal inhibitory concen-
tee for Clinical Laboratory Standards (5).                 trations of 2 or 4 ,ug/ml. The gentamicin-resis-
   Minimal inhibitory concentrations were determined

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by using a microtiter broth dilution method, which was     tant subpopulations gave LSI readings of 0.19 to
performed according to international collaborative         0.49, disk diffusion zone sizes of zero, and
study standards (2). Reference powders for each anti-      minimal inhibitory concentrations of 32 or 64 ,ug/
microbial agent tested were obtained from the drug         ml.
manufacturers.                                                These findings suggested that the discrepan-
   Separation of susceptible and resistant Pseudomonas     cies were due to random selection of the suscep-
subpopulations. Susceptible and resistant organisms        tible or resistant organisms in a mixed popula-
from populations of the seven Pseudomonas isolates         tion. The short incubation period with the
studied were separated by using the disk diffusion         Autobac I system might not allow the resistant
method. Standardized inocula containing 108 colony-
forming units per ml were streaked onto Mueller-           subpopulation in a mixed inoculum to grow to
Hinton plates on which disks containing 10 ,ug of          sufficient density for proper interpretation. To
gentamicin were placed. These plates were incubated        test this, the growth kinetics for the separated
at 35°C overnight. If double zones of inhibition were      subpopulations of Pseudomonas in the presence
observed, colonies within 10 mm of the disk and            of gentamicin elution disks were determined
colonies more than 15 mm from the disk were selected       during an extended incubation period by using
and tested individually by the Autobac I, disk diffu-      the Autobac I system. The results for a repre-
sion, and broth dilution methods.                          sentative isolate (isolate 33) are shown in Fig. 1.
   Growth kinetics of susceptible and resistant subpopu-   At 5 h, the susceptible strain had reached a
lations. The growth rates of the separated susceptible
and resistant strains of P. aeruginosa were determined     density sufficient for reading on the Autobac I
by using the Autobac I system. The organisms were          system. The maximum LSI of 1.0 and a corre-
incubated in Autobac I cuvettes in the presence of         sponding interpretation of susceptible persisted
gentamicin elution disks for up to 10 h, and LSI           during the entire incubation period. The resis-
readings were recorded at zero time and at hourly          tant strain did not grow to sufficient density for a
intervals.                                                 reading until after 6 h of incubation. LSI read-
   Growth kinetic studies were then conducted with         ings decreased during continued incubation and
mixtures of the susceptible and resistant subpopula-       gave interpretive readings of increased resist-
tions of the P. aeruginosa isolates. Each mixture was      ance during the incubation period. These results
prepared by combining 2 ml of each of the subpopula-
tions, which had been standardized individually with       demonstrated that the resistant strain grew more
the Autobac I system. Subsequent dilutions were            slowly than the susceptible strain and would not
made in Eugonic broth to yield final inoculum concen-      have been detected after 5 h of incubation.
trations of 106 to 2 x 106 colony-forming units per ml.       The growth rate of an experimental mixture of
The effect of extended Autobac I incubation on the         susceptible and resistant strains was determined
susceptibility results was determined as described         as described above for each Pseudomonas iso-
above. In addition, the relative numbers of susceptible    late. The Autobac I results for isolate 33 are
and resistant organisms in the mixture at zero time and    shown in Fig. 1. The mixture of susceptible and
at each incubation interval were determined. Samples
(100 ,uJ) were taken from the cuvettes, and serial 10-     resistant subpopulations could be read at 5 h,
fold dilutions were made in Mueller-Hinton broth; 0.1-     similar to the separated susceptible strain, and
ml samples were placed onto Mueller-Hinton plates          LSI values decreased during the extended incu-
with and without 10 ,g of gentamicin per ml. After 24      bation period, similar to the separated resistant
h the number of colonies on each plate was counted,        strain. The mixture was read by the Autobac I
and the proportions of susceptible and resistant organ-    system as susceptible at 5 h and resistant at 7.5
414       MAYO ET AL.                                                     ANTIMICROB. AGENTS CHEMOTHER.

 a) .8 S


      c3- R\

                                                                    1    2     3     4     5    6     7    8
      0                             8
   FIG. 1. Autobac I growth kinetics for susceptible        FIG. 2. Number of susceptible (0) and resistant
strains (O), resistant strains (A), and a mixture of      (A) organisms present during growth of a mixture of
                                                          the two strains of Pseudomonas isolate 33 when
susceptible and resistant strains (O) of Pseudomonas

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isolate 33 in the presence of gentamicin elution disks.   gentamicin was tested with the Autobac I method.
S, I, and R indicate the susceptible, intermediate, and   CFU, Colony-forming units.
resistant LSI breakpoints, respectively, for Autobac I
susceptibility determinations.
                                                          nas isolates indicated that mixed susceptible and
                                                          resistant populations might be a factor. Mixed
h. The resistant strain was not detected at the 5-        populations have been reported in Autobac I
h reading because of its slower growth.                   studies with cultures of methicillin-resistant S.
   More detailed growth curves were determined            aureus (1).
by removing samples from each cuvette and.                  Our results suggest that with Pseudomonas
counting the susceptible and resistant organisms          and gentamicin and possibly with other combi-
at hourly intervals. Figure 2 shows the results           nations, the prolonged lag phase of the resistant
for isolate 33. The resistant population de-              strains results in a failure of these strains to
creased during the first hour, entered a sustained        manifest themselves in a mixed culture during
lag period, and attained an exponential growth            the short incubation period required for Autobac
rate after 3 h. In contrast, the susceptible popu-        I susceptibility testing. Thus, a "false" reading
lation exhibited a brief lag period and rapidly           of susceptible could be obtained with the Auto-
entered the exponential growth phase. After 5 h,          bac I system, depending on the ratio of suscepti-
there was an approximate 2-log-greater number             ble strains to resistant strains selected for test-
of susceptible organisms present.                         ing. An extended period of incubation of the
   Subpopulations of susceptible and resistant            Autobac I cuvette has been suggested as a
organisms were separated from within popula-              remedy for the erroneous results obtained with
tions of the remaining six Pseudomonas-genta-             S. aureus and the penicillins (3). Our results also
micin discrepant isolates. Mixtures of the sepa-          indicate that a decrease in the LSI upon extend-
rated susceptible and resistant subpopulations            ed incubation of the cuvette could serve as a
for each isolate gave double zones of inhibition          marker for detecting such a mixture. However,
and decreased LSI values when the Autobac I               this requirement would compromise one of the
incubation period was extended.                           principle advantages of the Autobac I system,
                                                          the early determination and reporting of suscep-
                   DISCUSSION                             tibility results.
   Several factors have been proposed to explain             Another indicator of a mixed resistant and
discrepancies between the results obtained with           susceptible population could be an initial LSI of
the Autobac I and agar diffusion methods. These           <1.0. Funnell and Guinness (3) showed that
include differences in types of basal media,              their staphylococci that were methicillin suscep-
length of incubation, the narrow intermediate             tible, as determined by disk diffusion, invariably
range and greater sensitivity of the Autobac I            had LSIs of >0.9, whereas their staphylococci
system, and the technical and biological variabil-        that were methicillin resistant had LSIs of
ities inherent in comparisons between any two             <0.70. Six of our seven discrepant pseudomo-
test systems (7). In this study, the presence of          nads had initial LSIs between 0.6 and 0.97,
double zones of inhibition on agar diffusion              whereas 152 (96%) of our truly gentamicin-
plates with gentamicin-susceptible Pseudomo-              susceptible Pseudomonas sp. isolates exhibited
VOL. 21, 1982                              AUTOBAC I-DISK DIFFUSION DISCREPANCIES                                415

LSI readings of 1.0. Pseudomonas isolates ex-       allow an understanding of the nature of some of
hibiting LSIs of 0.6 to 0.9 for gentamicin might    the limitations of the Autobac I method.
contain mixed susceptible and resistant strains
and should be retested by conventional disk                             ACKNOWLEDGMENTS
diffusion, minimal inhibitory concentration           The assistance of the technologists of the Diagnostic Micro-
methods, or by extended incubation of the Auto-     biology Laboratory, Memorial Hospital, is gratefully acknowl-
bac I cuvette.                                      edged. Helen Rigopoulos was very helpful in the preparation
                                                    of the manuscript.
   In our original evaluation of the Autobac I
system, 42 significant discrepancies were ob-                             LITERATURE CITED
served during testing of 1,033 gram-negative        1. Cleary, T. G., and D. Maurer. 1978. Methicillin-resistant
isolates. The discrepancies which were of great-       Staphylococcus aureus susceptibility testing by an auto-
est concern from a clinical perspective, involv-       mated system, Autobac I. Antimicrob. Agents Chemother.
ing Pseudomonas and gentamicin, were studied           13:837-841.
in detail. We found that these isolates contained   2. Erlccson, H. M., and J. C. Sherris. 1971. Antibiotic sensi-
                                                       tivity testing: report of an international collaborative study.
subpopulations with different susceptibilities.        Acta Pathol. Microbiol. Scand. Sect. B. 217(Suppl.):1-90.
The other 35 discrepant organism-antibiotic         3. Funnell, G. R., and M. D. G. Guinness. 1979. Australian
combinations included Pseudomonas with car-            evaluation of Autobac I with suggested interpretive and
beniciilin and chloramphenicol, Escherichia and        technical modifications. Antimicrob. Agents Chemother.
Enterobacter with ampicillin, cephalothin, and      4. Harris, P. C., and L. B. Sealey. 1980. Two-hospital study
chloramphenicol, Serratia with carbenicillin and

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                                                       of Staphylococcus aureus susceptibility to penicillin and
chloramphenicol, Klebsiella with carbenicillin,        ampicillin by Autobac I. Antimicrob. Agents Chemother.
cephalothin, and gentamicin, and Proteus and           18:922-925.
                                                    5. National Committee for Clinical Laboratory Standards.
Citrobacter with cephalothin. In preliminary           1979. Performance standards for antimicrobial disc suscep-
studies, we found that 32 of these organisms also      tibility tests. Approved standard: ASM 2. National Com-
exhibited double zones of inhibition or de-            mittee for Clinical Laboratory Standards, Villanova, Pa.
creased LSIs after extended incubation when         6. Stubbs, K. G., and K. Wicher. 1977. Laboratory evaluation
                                                       of an automated antimicrobial susceptibility system. Am. J.
the particular antibiotic was tested.                  Clin. Pathol. 68:769-777.
   The Autobac I system interprets a dynamic        7. Thornsberry, C., T. L. Gavan, J. C. Sherris, A. Balows,
process by sampling a culture at a fixed interval      J. M. Matsen, L. D. Smith, F. Schoenknecht, L. D. Thrupp,
early in the growth curve. The results obtained        and J. A. Washington II. 1975. Laboratory evaluation of a
                                                       rapid automated susceptibility testing system: report of a
depend on the nature of the population and its         collaborative study. Antimicrob. Agents Chemother.
growth kinetics. Attention to these factors might      7:466-480.

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