Ampicillin Killing Curve Patterns of Haemophilus influenzae Type b

Transcription

1 ANTIROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1987, p /87/ $02.00/0 Copyright 1987, American Society for Microbiology Vol. 31, No. 11 Ampicillin Killing Curve Patterns of Haemophilus influenzae Type b Isolates by Agar Dilution Plate Count Method BERT F. WOOLFREY,* MARY E. GRESSER-BURNS, AND RICHARD T. LALLY Clinical Microbiology Section, Department of Anatomic and Clinical Pathology, St. Paul-Ramsey Medical Center, St. Paul, Minnesota Received 13 April 1987/Accepted 24 August 1987 The responses of 20 ampicillin-susceptible Haemophilus influenzae type b clinical isolates to the bactericidal action of ampicillin were studied by using a modified agar dilution plate count method. A well-defined paradoxical effect was observed in each of the 24-h killing curve patterns for 19 of the 20 isolates, the remaining isolate showing a less-well-defined but suggestive paradoxical effect after 48 h of ampicillin exposure. For each isolate, the lowest 24-h persister percentage representing maximum killing (paradoxical trough percentage) occurred over a narrow range of concentrations immediately above the, with such paradoxical trough percentages for the 20 isolates ranging from >0.1 to <0.001%. Three isolates selected to represent slow, intermediate, and rapid responses were investigated by repetition of 24-h studies and by determination of expanded killing curve patterns. Resultant agar dilution plate count killing curve patterns were found to be reproducible and strain dependent and served to characterize each isolate. The paradoxical effect became more distinct with the prolongation of ampicillin action. Maximum killing was again evident for a narrow range of ampicillin concentrations immediately above the, with persister percentages rising rapidly over the next few ampicillin concentrations to peak at 1 to 2 loglo increments higher than trough percentages. Based on the broad range of responses observed for the 20 isolates, the consistent presence of the paradoxical effect, and the time-dependent nature of bactericidal action, we suggest that the MBC and MBC/ ratios are inadequate indices of bactericidal action and that the all-or-none concept of "antimicrobial tolerance" should be abandoned. We recently described a novel agar dilution plate count (ADPC) method for the quantitative measurement of Staphylococcus aureus inhibition and killing by oxacillin (27-29). Since then, we have appropriately modified the method for application to Haemophilus influenzae and ampicillin. The present study investigates ampicillin inhibition and killing dynamics for H. influenzae type b isolates by using the modified ADPC method. MATERIALS AND METHODS Study design. A total of 20 recent cerebrospinal fluid ampicillin-susceptible H. influenzae type b isolates were randomly chosen from stock cultures. Upon retrieval from stock cultures, each isolate was reidentified as H. influenzae type b by standard microbiological methods and as ampicillin susceptible by agar dilution (AD) determination of the. Each isolate was then tested by the modified ADPC method to determine the, MBC, and 24-h ampicillin persister percentages. The resultant data were then used to compare the relationships between ADPC and AD s and to assess the appropriateness of using MBC/ ratios to identify slowly killed strains in accord with previously promulgated definitions of the so-called "antimicrobial tolerance" phenomenon. On the basis of the 24-h ampicillin persister percentage data indicating that H. influenzae type b strains constitute a spectrum ranging from some that are slowly killed to others that are rapidly killed, three representative isolates were selected for further study to determine the reproducibility and potential strain dependency of 24-h persister percentages and to investigate the characterization of H. influenzae type b strains by using expanded * Corresponding author. killing curve patterns determined after various sequential exposures to ampicillin. Isolates. A total of 20 ampicillin-susceptible H. influenzae type b isolates were randomly selected from the St. Paul- Ramsey Medical Center Clinical Microbiology Laboratory stock culture collection, representing a large number of recently acquired cerebrospinal fluid isolates from cases of meningitis throughout Minnesota. The stock isolates were maintained in defibrinated sheep blood at -70 C and retrieved by two 24-h passages on chocolate agar incubated in 5% CO2 at 35 C. AD susceptibility tests. AD s were determined on chocolate Mueller-Hinton agar for each of the 20 isolates by using a replicator method in accordance with the recommendations of the National Committee for Clinical Laboratory Standards (18). ADPC tests. (i) General modifications. For testing H. influenzae and ampicillin, the ADPC method (27-29) was modified by the use of the following: MHB-C (Mueller- Hinton broth [Difco Laboratories, Detroit, Mich.] containing 1% supplement C [Difco]) and MHA-C (Mueller-Hinton agar II [BBL Microbiology Systems, Cockeysville, Md.] containing 1% supplement C) media to promote and support the growth of H. influenzae; a standardized method for inoculum preparation to ensure consistent densities of exponential-phase cells; quantitative surface streak inoculation of ADPC plates to circumvent the thermal degradation of inoculum density as experienced using pour plate preparations of H. influenzae; ampicillin inactivation by a 1:10 aqueous dilution of Penase (Difco); and a humidified 5% CO2 atmosphere for plate incubation. (ii) Inoculum preparation. Material from 8 to 10 typical H. influenzae type b colonies on the second stock retrieval plate was used to initiate 5-ml MHB-C cultures in borosilicate glass tubes (16 by 125 mm; American Scientific Products, 1711

2 1712 WOOLFREY ET AL. McGaw Park, Ill.). Each culture tube was incubated in air at 35 C with continuous agitation, and growth was closely monitored until the visual density approximated that of a 0.5 McFarland standard, this procedure having previously been demonstrated by us to produce exponential-phase cultures with densities consistently approximating 3 x 108 CFU/ml. Each culture was then further diluted as needed with MHB- C to produce standardized inoculum preparations for the ADPC tests. For each inoculum preparation the actual reference cell density was determined as follows. Onto the surface of each of two 100-mm non-ampicillin-containing MHA-C plates, 0.1 ml of an appropriate dilution of the inoculum preparation was pipetted and quantitatively streaked by thorough dispersion in at least three opposing directions by using a bacteriologic loop to cover the entire surface. The streaked plates were allowed to dry for approximately 15 min, overlaid with 10 ml of non-ampicillincontaining 50 C molten MHA-C, and incubated at 35 C in a humidified 5% CO2 atmosphere for 24 h. Colony counts were then performed for calculation of the actual inoculum density. (iii) Preparation and inoculation of ADPC panels. On the day of use, ADPC plates were prepared with MHA-C to contain ampicillin (Bristol Laboratories, Syracuse, N.Y.) in twofold dilution concentrations ranging from 0.12 to 256,ug/ml. Each plate of a dilution panel was inoculated by pipetting 0.05 ml (-1.5 x 105 CFU per plate) of a standardized inoculum preparation onto the agar surface with immediate quantitative streaking, as described above, to disperse the inoculum evenly. Inoculated plates were allowed to dry for approximately 15 min, at which time they were overlaid with 10 ml of molten MHA-C containing an analogous ampicillin concentration. Panels were prepared in duplicate for each isolate and incubated in a humidified 5% CO2 atmosphere at 35 C. (iv) Determination of the, MBC, and 24-h persister percentages. ADPC panels were removed from incubation at 24 h, at which time plate counts were performed to determine s. The was defined as the ampicillin concentration in the first plate of the ascending concentration series for which the colony count was at least two standard deviations below that representing 0.1% of the actual inoculum reference count. In actual practice, colony counts on the plate and at higher concentrations were virtually absent even when plates were incubated for prolonged periods of time. Upon completion of the plate counts, each plate was overlaid with 1 ml of a 1:10 dilution of Penase in sterile deionized H20 to inactivate ampicillin. The plates were then incubated for 48 h for the development of 24-h persister plate counts. The MBC was defined as the ampicillin concentration in the first plate of the ascending concentration series for which the plate count was at least two standard deviations below that representing 0.1% of the inoculum reference count, with counts of.0.1% being permitted for any or all of the higher concentrations. In addition, the persister percentage was calculated for each ampicillin concentration as the percentage of the actual inoculum showing regrowth as CFU. The paradoxical or Eagle effect (5) was defined as the occurrence of progressively increasing plate counts for at least three consecutive ampicillin concentrations above the. For purposes of later presentation and discussion, the terms "paradoxical trough" and "paradoxical peak" persister percentages will be used to indicate the lowest and highest percentages of surviving inoculum encountered, respectively, in the biphasic paradoxical effect for ascending ampicillin concentrations above the. ANTIROB. AGENTS CHEMOTHER. (v) Expanded ADPC killing patterns. ADPC panels were prepared and inoculated as described above for and MBC testing. For each isolate, a sufficient number of ADPC panels were prepared so that duplicate panels could be overlaid with Penase solution to inactivate ampicillin at appropriate times for the determination of killing curves. For isolate 254, the most slowly killed isolate of the 20 studied, ampicillin was inactivated at 12, 24, 48, and 72 h. For isolate 356, representing isolates with intermediate responses, ampicillin was inactivated at 6, 12, 24, and 48 h. For isolate 914, the most rapidly killed isolate of the 20 studied, ampicillin was inactivated at 3, 6, 12, and 24 h. After Penase application, the duplicate panels were incubated for 48 h, at which point colony counts were made to determine persister percentages for each ampicillin concentration. For each of the three isolates selected for this portion of the study, strain dependency and killing curve reproducibility were assessed by repeating the 24-h ADPC tests with newly retrieved stock. (vi) Quality control. Initial ADPC tests on the H. influenzae type b isolates showed that rapid and sometimes complete inoculum degradation occurred during the pour plate preparation step. It was found that inoculum degradation did not occur if the pour plate inoculation step was replaced by quantitative surface streaking followed by an overlay of 10 ml of 50 C molten MHA-C. For isolates 254, 356, and 914, whose killing curve patterns are presented in the figures, as well as for other selected isolates, actual inoculum density calculations made from companion overlaid and nonoverlaid preparations showed no evidence of inoculum degradation due to the molten agar overlay. Because ampicillin may be unstable under various conditions (20, 24), including its use with supplement C (26), and because ampicillin action times were longer than 24 h for some of the expanded ADPC tests, we performed the following studies to determine the extent of ampicillin inactivation. MHA-C ampicillin AD panels were prepared on the day of use. Using the AD replicator method (18), we inoculated duplicate panels with Escherichia coli ATCC and S. aureus ATCC after 0, 24, 48, and 72 h of incubation at 35 C and performed separate trials in air and in a humidified 5% CO2 atmosphere. After inoculation, panels were incubated for 24 h, and s were determined as the lowest ampicillin concentration allowing no growth. For the two organisms, s at 24 h after inoculation at zero time were within the known quality control limits. With each additional 24-h incubation of plates prior to inoculation, s for each organism increased approximately one twofold dilution step. Because the 24-h s for the organisms were within reference quality control limits determined in non-supplement C-containing media, it was concluded that the apparent loss of ampicillin activity was caused by prolonged incubation rather than P-lactamase activity associated with supplement C. This demonstrated loss of ampicillin activity must therefore be considered in the interpretation of killing curve patterns representing prolonged ampicillin exposure. For each ADPC trial, internal quality control was provided by comparing the ADPC result with the reference AD result for each H. influenzae type b isolate. For all trials, as discussed below, the ADPC was found to be equal to or less than the corresponding AD. RESULTS ADPC s determined after 24 h of incubation were consistently lower than companion AD s, with approx-

3 VOL. 31, 1987 H. INFLUENZAE KILLING CURVE PATTERNS A B h h 4P ) ' Y i X Y Q&E \ai 48h \,O/.V.v.v i jug / ml Sg / ml FIG. 1. (A) Ampicillin 24-h killing curve patterns for isolate 254 as determined on three separate trials. The ordinate represents the percentage of inoculum persisting as viable CFU after 24 h of ampicillin action. The abscissa represents ampicillin concentrations (micrograms per milliliter) in ADPC plates. 0, 21,000 CFU. (B) Expanded ampicillin killing curve patterns for isolate 254. The ordinate represents the percentage of inoculum persisting as viable CFU. The abscissa represents ampicillin concentrations (micrograms per milliliter) in ADPC plates. Curves designated 12, 24, and 48 h represent the percentages of inoculum persisting as viable CFU after the indicated duration of ampicillin action. The dashed and solid lines for 48 h represent two separate trials. 0,.1,000 CFU. imately 5% being the same and with 48, 42, and 5% being one concentration step, two steps, and three steps lower, respectively. Postponement of the ADPC reading to 48 h produced a twofold dilution step increase for only an occasional isolate. For the 20 isolates, 19 exhibited MBC/ ratios of c8, with the majority having ratios of 1 or 2. Only one isolate (254) had a higher MBC/ ratio (.1,024), making it the single isolate to qualify as ampicillin tolerant according to the commonly used definition (MBC/ ratio,.32). Killing curve patterns determined after 24 h of ampicillin action showed that 19 of the 20 isolates exhibited paradoxical effects. The single isolate (254) which did not exhibit a distinct paradoxical effect in the 24-h killing curve did exhibit the suggestion of paradoxical patterns after longer ampicillin action times (48 and 72 h). Paradoxical trough 24-h persister percentages for the 20 isolates were distributed within 0.5 log10 increments as follows: 2 ( to <0.001%), 6 (0.001 to <0.005%), 1 (0.005 to <0.01%), 6 (0.01 to <0.05%), 4 (0.05 to <0.1%), and 1 (0.1 to <0.5%). Overall, paradoxical trough percentages occupied a relatively narrow range of ampicillin concentrations (one to eight times the ), with the majority exhibiting a narrower range (two to four times the ). Beyond the trough ampicillin concentrations, persister percentages increased rapidly to reach peak percentages at two to three twofold dilution steps higher. - \-V.- The reproducibility and strain dependency of 24-h killing curve patterns are shown in Fig. 1A, 2A, and 3A for isolates 254, 356, and 914, respectively, which were selected to illustrate the range of responses to 24-h ampicillin action encountered for the 20 isolates. The several curves plotted for each isolate represent separate independent trials with stock cultures and illustrate the reproducibility of the patterns. A comparison of the patterns for the three isolates reveals strain dependency, with persister percentages at two to four times the separating the strains by differences of approximately 1 to 2 logl0 steps. For isolate 254 (Fig. 1A), >0.1% of the inoculum survived 24 h of ampicillin action for all concentrations tested, with no demonstrable paradoxical peak. For this isolate however, the paradoxical effect was suggested with prolonged ampicillin action beyond 24 h, as discussed below. For isolate 356 (Fig. 2A) and isolate 914 (Fig. 3A), the 24-h killing curve patterns again demonstrated the narrow range of ampicillin concentrations for the paradoxical trough and the rapid increase in persister percentages to reach peak values greatly exceeding the 0.1% breakpoint commonly used in MBC determinations. Figures 1B, 2B, and 3B represent expanded killing curve patterns for isolates 254, 356, and 914, respectively. For each isolate, each curve represents a given duration of ampicillin action. For isolate 254, persister percentages for 12 and 24 h of ampicillin action remained significantly above

4 1714 WOOLFREY ET AL. ANTIROB. AGENTS CHEMOTHER. B U) 24h 0.01X v v as 266 g / ml g /ml FIG. 2. (A) Ampicillin 24-h killing curve patterns for isolate 356 as determined on four separate trials. The ordinate and abscissa are as in the legend to Fig. 1A. 0, 21,000 CFU. (B) Expanded ampicillin killing curve patterns for isolate 356. The ordinate and abscissa are as in the legend to Fig. 1B. Curves designated 6, 12, 24, and 48 h represent the percentages of inoculum persisting as viable CFU after the indicated duration of ampicillin action. 0, -1,000 CFU. 0.1%, showing no definite paradoxical pattern. After 48 h of ampicillin action, as can be seen from the plots of independent trials, persister percentages continued to decrease, with the patterns now suggesting the appearance of the paradoxical effect. After 72 h of incubation (pattern not plotted), persister percentages were only slightly lower than those at 48 h, indicating almost complete exhaustion of ampicillin action. Figure 2B shows the expanded killing curve patterns for isolate 356, representing those isolates which were killed by ampicillin at an intermediate rate. After 12 h of ampicillin action, paradoxical trough percentages approached 0.5%, decreasing to approximately 0.03% after 24 h. Paradoxical peaks became prominent with time, exceeding 2 logl0 steps above trough values after 48 h, with peak percentages remaining above 0.1% for the higher concentrations. Figure 3B shows the expanded killing curve patterns for isolate 914, which was the most rapidly killed isolate of the 20 studied. Paradoxical peaks again became more prominent with time, with peak percentages remaining above 0.1% at 24 h. DISCUSSION We recently introduced the ADPC method (27-29) as an alternative to the broth dilution plate count (BDPC) approach for the quantitative measurement of bacterial inhibition and killing by P-lactam agents. The procedure was designed to avoid the severe dependency on technical factor 418h variations which have been shown to confound BDPC results. This was done for ADPC by immobilization of the inoculum in an agar-gel matrix, both throughout the time of bacterial exposure to the antimicrobial agent and during the time for regrowth of viable CFU after inactivation of the agent. ADPC thus minimized the introduction of spurious measurements due to bacteria which remain viable by means of "adaptive" mechanisms such as those which might protect microorganisms from the action of the agent (cell clumping, capsule and glycocalyx production, and inadvertent sequestration of bacteria on tube walls above broth surfaces), the induction of slow cell growth or cell dormancy (ph drop, nutritional depletion, and the use of nonexponentially growing or stationary-phase inoculum preparations), and the carry-over of inhibitory amounts of antimicrobial agent onto MBC plates. Thus, for a given antimicrobial agent, concentration, and time of action, ADPC measurements should largely represent microorganisms which remain viable on the basis of "intrinsic" properties genetically determined. Studying S. aureus and oxacillin by ADPC, we found responses to be strain dependent, with strains constituting a spectrum ranging from some which are slowly killed to others which are rapidly killed (29). The paradoxical effect (5), although irregularly observed by BDPC methods, was observed by us for all S. aureus isolates tested, with the phenomenon becoming manifest over the course of antimicrobial action. On the basis of the killing curve patterns

5 VOL. 3 1, 1987 A H. INFLUENZAE KILLING CURVE PATTERNS B h , cn (a 4-, cca! C- 4-' a! jug / ml i jig / ml FIG. 3. (A) Ampicillin 24-h killing curve patterns for isolate 914 as determined on four separate trials. The ordinate and abscissa are as in the legend to Fig. 1A. 0,.1,000 CFU. (B) Expanded ampicillin killing curve patterns for isolate 914. The ordinate and abscissa are as in the legend to Fig. 1B. Curves designated 3, 6, 12, and 24 h represent the percentages of inoculum persisting as viable CFU after the indicated duration of ampicillin action. *,.1,000 CFU. observed for S. aureus and oxacillin, we concluded that the MBC and MBC/ ratios are arbitrary, artificial, and invalid concepts for the characterization of bactericidal action and that the all-or-none concept of tolerance as conceived by most clinical investigators should be abandoned. There is a paucity of information available on the bactericidal action of ampicillin or similar agents against exponential-phase cultures of H. influenzae. Bergeron and Lavoie (1) described ampicillin tolerance (MBC/,.32) for 9 of 165 ampicillin-susceptible strains tested as stationary-phase inoculum preparations. When the nine were subsequently tested as exponential-phase inoculum preparations, only four remained tolerant. Tolerance was described as being rapidly lost at room temperature storage but as being preserved for stock cultures at -70'C. With a single ampicillin concentration of 2 F±g/ml, killing curve patterns determined at 2, 6, 12, and 24 h were said to show survivor percentages which correlated well with MBCs. However, straindependent killing rates were' not sharply defined, and the paradoxical effect could not be observed because of the use of'a single ampicillin concentration. Because of the known unreliability of BDPC results, we studied the response of H. influenzae type b strains to ampicillin by using ADPC modified to compensate for the sensitive and fastidious nature of the microorganism. Similar to our observations with S. aureus and oxacillin, we found the H. influenzae type b response to be strain dependent, with strains again constituting a spectrumn ranging from some which are slowly killed to others which are more rapidly killed. A distinct paradoxical effect was found after 24 h of ampicillin action for 19 of the 20 isolates studied, the exception being the most slowly killed isolate which, after 48 h of ampicillin action, developed a pattern that did not fit our rigid definition but that approximated the paradoxical effect. Although the observed diminished paradoxical response may represent a characteristic of the slowly killed isolate, a probable alternative explanation may be found in the dynamics of a slowly killed isolate exposed to significantly decaying ampicillin activity. Similar to our observations with S. aureus and IH. influenzae type b, strain-dependent killing by 13-lactam agents'has recently been described for streptococci. Jokipii and colleagues (13), using BDPC methods to study group B streptococci, found that concentrations immediately above the exerted maximum bactericidal activity on exponentialphase inoculum preparations, with time kill studies qxtending over 24 h showing distinct strain differences. They concluded that the dynamic nature of bactericid,al activity could not be meaningfully reduced to one measurement such as that of the MBC. Meylan and colleagues (16) found reproducible strain-dependent killing for strains of Streptococcus sanguis and Streptococcus mitis using 24 h of amoxicillin action. Strains of S. sanguis were killed on the average much more slowly'than strains of S. mitis, with strain-dependent killing demonstrated for strains of each species. Although the maximum killing response was de-

6 1716 WOOLFREY ET AL. scribed for a narrow range of concentrations immediately above the, the paradoxical effect was not said to be consistently observed. For the strains, relative killing determined at 2 and 4 h was said to correspond well with that observed after 24 h, the latter results showing a regular distribution of strains that was not detected by MBC measurements and correlated poorly with tolerance as determined by MBC/ ratios. Despite much effort by many investigators, the biochemical mechanisms responsible for differences in bacterial killing are yet to be determined. Although the slow killing and lysis of some mutant bacterial strains, such as the Streptococcus pneumoniae mutant described by Tomasz and colleagues (23, 25), have been linked to the production of autolysin inhibitors resembling lipoteichoic acids, it has not been established that similar mechanisms may be responsible for the spectrum of strain-dependent differences now established for other bacteria. A consistent observation since the early studies on penicillin action has been that the response for a given in vitro system is directly related to the rapidity of cell growth, and evidence has been presented that individual cell responses may differ greatly depending upon the stage of the cell cycle when exposed to an agent (3, 12, 22). On this basis, strain-dependent differences in responses might be expected if similarly prepared exponentially growing inoculum preparations contained different genetically determined proportions of responsive cells, either in various stages of their metabolic cycles or having minor differences in growth rates that are expressed as significant differences in killing dynamics. On the basis of the observations of Hartmann and colleagues (11), Greenwood (7) postulated that cells with no apparent requirement for hydrolase activity and thus completed envelopes might be placed in a state of suspended animation by higher concentrations of penicillin. Recent observations made on strains of streptococci exposed to low concentrations of penicillin known to optimally inhibit the assembly of insoluble peptidoglycan suggest that responses may be related to the extent by which RNA and protein syntheses lag the inhibition of peptidoglycan synthesis (17, 22). With this arrangement, it was postulated that penicillin may act principally as a static agent directed towards nonlethal penicillin-binding protein targets, with death occurring only as a secondary or tertiary phenomenon mediated by molecular feedback mechanisms that fail to inhibit lethal activities related to continued RNA and protein syntheses. For any system designed to study bactericidal activity, the roles of protoplasts and spheroplasts as causes of apparent slow bacterial killing and persistence must also be considered. As described for both S. aureus (10) and H. influenzae (2, 19), protoplasts and spheroplasts may be produced under conditions found in commonly used broth and agar cultures. Studies involving parallel observations on bacterial morphogenesis, microscopic enumeration, and cell viability for susceptible bacteria exposed to,-lactam agents (4, 6, 10, 14, 15, 30) have shown that morphologic alterations compatible with cell wall-deficient forms, loss of viability, decreases in cell count, and lysis occur sequentially within a few hours at concentrations immediately above the, whereas prolonged viability, minimum morphologic changes, and lack of lysis occur at concentrations significantly above the. The percentage of such cell forms which might be resuscitated and thus included as a component of the viable cells surviving antimicrobial agent action remains problematic. However, observations by other investigators (8-10, 21) suggest that cell wall-deficient forms play little if any role in ANTIROB. AGENTS CHEMOTHER. bacterial survival and persistence in vitro. Although the ADPC method was designed to avoid the induction and incorporation of adaptively protected bacteria into the measurements, the potential inclusion of revivable cell walldeficient forms exists and needs to be studied. The most appropriate terminology to be used for bacteria which remain viable after exposure to an antimicrobial agent in vitro is debatable. We have previously (27-29) used persisters as a general term to include microorganisms which remain viable through adaptive as well as intrinsic mechanisms, the latter being manifest for any given in vitro system as reproducible strain-dependent responses which are presumably genetically determined. For accurate and precise quantitation of intrinsic persisters, useful test systems must avoid the incorporation of adaptive persisters, and measurements of intrinsic persisters must be qualified by the inclusion of data indicating the antimicrobial agent used, the concentration of the agent, the length of exposure to the agent, the phase and density of the inoculum, and a precise determination of the inoculum percentage remaining viable. Additionally, optimum characterization of the response of any strain requires the use of an extended range of both antimicrobial concentrations and times of antimicrobial action to demonstrate the presence and amplitude of the paradoxical effect. In this way, strains may be compared as to their relative responses rather than being judged as tolerant or nontolerant on the basis of artificial breakpoints. The relative nature of the strain-dependent response and the apparent universal presence of the paradoxical effect must clearly be kept in mind for any future study designed to determine whether differences in bactericidal activity may in any way be related to the clinical effectiveness of antimicrobial agents. ACKNOWLEDGMENTS This research was supported by grants and from the St. Paul-Ramsey Foundation. LITERATURE CITED 1. Bergeron, M. G., and G. Y. Lavoie Tolerance of Haemophilus influenzae to f-lactam antibiotics. Antimicrob. Agents Chemother. 28: Bottone, E. J., Z. Brandman, and S. S. Schneierson Spheroplasts of Haemophilus influenzae induced by cell wallactive antibiotics and their effect upon the interpretation of susceptibility tests. Antimicrob. Agents Chemother. 9: Chain, E., and E. S. Duthie Bactericidal and bacteriolytic action of penicillin on the staphylococcus. Lancet i: Ciak, J., and F. E. Hahn Concurrent morphological and chemical events in Staphylococcus aureus exposed to penicillin. Science 137: Eagle, H., and A. D. Musselman The rate of bactericidal action of penicillin in vitro as a function of its concentration, and its paradoxically reduced activity at high concentrations against certain organisms. J. Exp. Med. 88: Eriksen, K. R Some studies on the lytic action of penicillin on staphylococci and pneumococci. Acta Pathol. Microbiol. Scand. 23: Greenwood, D Mucopeptide hydrolases and bacterial "persisters." Lancet ii: Greenwood, D., and F. O'Grady Trimodal responses of Escherichia coli and Proteus mirabilis to penicillins. Nature (London) 228: Gunnison, J. G., M. A. Fraher, and E. Jawetz Persistence of Staphylococcus aureus in penicillin in vitro. J. Gen. Microbiol. 35: Hamburger, H., and J. Carleton Staphylococcal spheroplasts as persisters, p In L. B. Guze (ed.), Microbial

7 VOL. 31, 1987 protoplasts, spheroplasts and L-forms. The Williams & Wilkins Co., Baltimore. 11. Hartmann, R., J. Holtje, and U. Schwarz Targets of penicillin action in Escherichia coli. Nature (London) 235: Holtzhoffer, S., R. Sussmuth, and R. Haag Oscillating tolerance in synchronized cultures of Staphylococcus aureus. Antimicrob. Agents Chemother. 28: Jokipii, L. A., P. Brander, and A. M. M. Jokipii Reverse inoculum effect in bactericidal activity and other variables affecting killing of group B streptococci by penicillin. Antimicrob. Agents Chemother. 27: Klein, R. D., and G. H. Luginbuhl Ampicillin-induced morphological alterations of Haemophilus influenzae type b. Antimicrob. Agents Chemother. 11: Lederberg, J., and J. St. Clair Protoplasts and L-type growth of Escherichia coli. J. Bacteriol. 75: Meylan, P. R., P. Francioli, and M. P. Glauser Discrepancies between MBC and actual killing of viridans group streptococci by cell-wall-active antibiotics. Antimicrob. Agents Chemother. 29: Mychajlonka, M., T. D. McDowell, and G. D. Shockman Inhibition of peptidoglycan, ribonucleic acid, and protein synthesis in tolerant strains of Streptococcus mutans. Antimicrob. Agents Chemother. 17: National Committee for Clinical Laboratory Standards Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M-7A, December, National Committee for Clinical Laboratory Standards, Villanova, Pa. 19. Roberts, D. E., A. Ingold, S. V. Want, and J. R. May Osmotically stable L forms of Haemophilus influenzae and their significance in testing sensitivity to penicillins. J. Clin. Pathol. 27: Ryan, K. J., G. M. Needham, C. L. Dunsmoor, and J. C. Sherris Stability of antibiotics and chemotherapeutics in agar plates. Appl. Microbiol. 20: Seeberg, S., and J. E. Brorson Prolonged survival of H. INFLUENZAE KILLING CURVE PATTERNS 1717 non-multiplying Staphylococcus aureus cells undergoing autolysis in the presence of certain inhibitors of cell wall synthesis. Scand. J. Infect. Dis. 7; Shockman, G. D., L. Daneo-Moore, T. D. McDowell, and W. Wong Function and structure of the cell wall-its importance in the life and death of bacteria, p In M. R. J. Salton and G. D. Shockman (ed.),,-lactam antibiotics. Academic Press, Inc., New York. 23. Tomasz, A., A. Albino, and E. Zanati Multiple antibiotic resistance in a bacterium with suppressed autolytic system. Nature (London) 227: Trissel, L. A Handbook on injectable drugs, 3rd ed., p American Society of Hospital Pharmacists, Bethesda, Md. 25. Waks, S., and A. Tomasz Secretion of cell wall polymers into the growth medium of lysis-defective pneumococci during treatment with penicillin and other inhibitors of cell wall synthesis. Antimicrob. Agents Chemother. 13: Washington, J. A., II, R. J. Snyder, and P. C. Kohner Spurious ampicillin resistance by testing Haemophilus influenzae with agar containing supplement C. Antimicrob. Agents Chemother. 9: Woolfrey, B. F., R. T. Lally, and M. N. Ederer Evaluation of oxacillin tolerance in Staphylococcus aureus by a novel method. Antimicrob. Agents Chemother. 28: Woolfrey, B. F., R. T. Lally, and M. N. Ederer Influence of technical factor variations during inoculum preparation on the agar dilution plate-count method for quantitation of Staphylococcus aureus oxacillin persisters. Antimicrob. Agents Chemother. 30: Woolfrey, B. F., R. T. Lally, M. N. Ederer, and M. Gresser- Burns Oxacillin killing curve patterns of Staphylococcus aureus isolates by agar dilution plate count method. Antimicrob. Agents Chemother. 31: Yourassowsky, E., M. P. Van der Linden, M. J. Lismont, F. Crokaert, and Y. Glupczynski Effect on growth curve patterns of brief exposure of bacteria to different concentrations of P-lactam antibiotics. J. Antimicrob. Chemother. 15(Suppl. A): Downloaded from on October 31, 2018 by guest