ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1991, p. 1824-1828 0066-4804/91/091824-05$02.00/0

Vol. 35, No. 9

Copyright ©D 1991, American Society for Microbiology

Bactericidal Effects of Antibiotics on Slowly Growing and Nongrowing Bacteria ROBERT H. K. ENG,1* FRANK T. PADBERG,2 SHARON M. SMITH,3 ELIZABETH N. TAN,4 AND CHARLES E. CHERUBIN4 Infectious Disease,' Vascular Surgery,2 and Microbiology3 Sections, Department of Veterans Affairs Medical Center, East Orange, New Jersey 07019; Departments of Medicine,' Surgery,2 and Pathology and Microbiology,3 New Jersey Medical CollegelUMDNJ, Newark, New Jersey 07103; and Infectious Disease Research Institute, Jericho, New York 117534 Received 2 April 1991/Accepted 12 July 1991

Antimicrobial agents are most often tested against bacteria in the log phase of multiplication to produce the maximum bactericidal effect. In an infection, bacteria may multiply less optimally. We examined the effects of several classes of antimicrobial agents to determine their actions on gram-positive and gram-negative bacteria during nongrowing and slowly growing phases. Only ciprofloxacin and ofloxacin exhibited bactericidal activity against nongrowing gram-negative bacteria, and no antibiotics were bactericidal (3-order-of-magnitude killing) against Staphylococcus aureus. For the very slowly growing gram-negative bacteria studied, gentamicin (an aminoglycoside), imipenem (a carbapenem), meropenem (a carbapenem), ciprofloxacin (a fluoroquinolone), and ofloxacin (a fluoroquinolone) exhibited up to 5.7 orders of magnitude more killing than piperacillin or cefotaxime. This is in contrast to optimally growing bacteria, in which a wide variety of antibiotic classes produced 99.9% killing. For the gram-positive and gram-negative bacteria we examined, antibiotic king was greatly dependent on the growth rate. The clinical implications of slow killing by chemotherapeutic agents for established bacterial infections and infections involving foreign bodies are unknown.

The effectiveness of an antimicrobial agent is measured by its ability to inhibit and kill bacteria. However, tests of this ability are usually performed on log-phase bacteria or those in a very rapid-growth phase in media supplying all of the necessary nutrients for optimal growth (11). Clinically, microorganisms in some infected tissues may be walled off quickly by host leukocytes, followed by fibrin deposits, and thus the growth of the bacteria may be less optimal and controlled by the limited access to nutrition (13). Other evidence for the importance of studying dormant organisms is available. For Staphylococcus epidermidis, Widmer et al. showed that only data on antibiotic killing of static organisms correlated with successful therapy (16). Isolates of S. aureus from chronic subcutaneous cage infections in rats were shown to be in a state of dormancy (4). In these scenarios, the organisms multiply at a less-than-optimal rate and the effectiveness or lack of effectiveness of antibiotics against organisms in such a growth state is just being recognized. It is well known that a P-lactam antibiotic exerts its maximum effect on rapidly growing bacteria, but the limits of its activities against nongrowing and slowly growing bacteria need to be defined. MATERIALS AND METHODS Bacterial strains and antibiotics. The bacterial strains used included clinical strains of Klebsiella pneumoniae, Serratia marcescens, and Enterobacter cloacae and the American Type Culture Collection organisms Staphylococcus aureus 25913, Pseudomonas aeruginosa 27853, and Escherichia coli 25922. All of the antibiotics used were obtained from their respective manufacturers and diluted as recommended prior to use. Preparation of the inoculum. Log-phase bacteria were *

Corresponding author.

prepared from an overnight growth of colonies on sheep blood tryptic soy agar after they had been thawed from storage in a -70°C freezer. Three to four colonies were inoculated into optimal broth (as defined below for optimalgrowth studies) or suboptimal broth (as defined below for suboptimal-growth studies), and after 4 h of incubation at 35°C, the organisms were diluted with the respective prewarmed liquid culture media to the desired inoculum concentration. Stationary-phase bacteria were grown for 24 h in Mueller-Hinton broth and diluted in warm sterile limiting growth media to the desired inoculum. A high inoculum concentration of 108 CFU/ml was used, similar to the high inoculum concentration used in a chemostat culture of P. aeruginosa with carbapenems (17). This high inoculum concentration has to be used to avoid the antibiotic carry-over effect (8) when using antibiotics in the achievable blood concentrations, as suggested by the National Committee for Clinical Laboratory Standards (10). Media controlling rate of growth. The basic media used were different for gram-positive and gram-negative organisms. For gram-negative bacteria, the three media used were M9 (12) with no glucose carbon source added and with a 0.05% or 0.5% (wt/vol) glucose carbon source added. These media support no growth, slow growth, and rapid growth, respectively. For gram-positive bacteria, we used yeast nitrogen base broth alone, with an amino acid solution and a 0.05% glucose carbon source, and with. a Casamino Acids (17 g/liter; Difco Laboratories, Detroit, Mich.) solution and a 0.5% glucose carbon source added. These three media support no growth, suboptimal growth, and optimal growth, respectively. Susceptibilities to the antibiotics were determined for gram-positive and gram-negative bacteria in the respective media and in Mueller-Hinton broth by using the microtiter method and a standard inoculum concentration of 5 x 105 CFU/ml. Killing kinetics. At zero time, the static-phase inoculum 1824

VOL. 35, 1991

was added to an equal volume of no-growth medium to a final density of 108 CFU/ml in 5 ml containing either no antibiotic or the desired antibiotic concentration (see Table 2). The antibiotic concentrations are those usually achieved in the blood during treatment as suggested by the National Committee for Clinical Laboratory Standards (10). For the carbapenems, nafcillin, cefotaxime, and piperacillin, the concentrations used were 5 to 25 pug/ml; the quinolones were used at 4 ,ug/ml; gentamicin was used at 8 ,ug/ml; and rifampin was used at 1 Vug/ml. At 0, 3, 6, and 24 h of incubation at 35°C, a portion of each incubation mixture was subcultured quantitatively onto sheep blood agar and the number of viable colonies was determined at 24 h of incubation of the plates. Each experiment was performed twice on separate days to ensure congruence of results. Optical density measurements. The optical densities of the incubation mixtures were measured at the time of subculture. Optical density was measured with a path length of 1 cm at 600 nm by using a 300-N spectrophotometer (Gilford Laboratories, Oberlin, Ohio).

RESULTS Control of the rate of bacterial growth. For gram-negative bacteria, the limiting factor for growth was the amount of the carbon source. Thus, controlling the initial amount of carbon nutrients controlled the growth rate. An example of this is shown in Fig. 1A for E. coli ATCC 25922. Other E. coli strains grew at the same rates as the strain shown. For gram-positive bacteria, the organisms need thiamine, nicotinic acid, and amino acids, as well as a carbon source. The growth rate can be controlled by reducing the carbon and nitrogen sources, as demonstrated for S. aureus ATCC 29213 in Fig. 1B. Other strains of S. aureus grew at the same rates in the respective media as the strain shown. The MICs of the antibiotics for the organisms studied are presented in Table 1. The media designed to control growth rate did not affect susceptibility to the antibiotics, except for gentamicin, whose MICs were higher in all of the media than in MuellerHinton broth, possibly because of divalent cation interaction with this drug. Killing of bacteria in the nongrowing phase. The representative antimicrobial agents of classes usually thought to be bactericidal were tested against gram-positive and gramnegative organisms. Concentrations of antibiotics achievable in serum or near those achievable in serum were used against an inoculum density of 108 CFU/ml. Among the antimicrobial agents tested were gentamicin, nafcillin, cefotaxime, piperacillin, imipenem, meropenem, ciprofloxacin, ofloxacin, and rifampin. Only the two fluoroquinolones were able to produce any bactericidal effects against nongrowing gramnegative bacteria (Table 2). Gentamicin was notably inactive against most of the bacteria tested in the absolute nongrowing phase. For nongrowing S. aureus, no antibiotic was really bactericidal (Table 3). Besides this S. aureus strain, we also investigated three clinical strains resistant only to penicillin and found the same lack of killing by antibiotics other than the quinolones. Killing of slowly growing bacteria. When 1/10 of the carbon source was provided, gram-negative bacteria grew suboptimally. Among the antibiotics tested, several showed good activity. Gentamicin, the two carbapenem antimicrobial agents, and the fluroquinolones produced the best killing responses. The rank order of killing of gram-negative bacteria was generally gentamicin, followed by the two fluoroquinolones, followed by the two carbapenems, which produced

BACTERICIDAL EFFECTS OF ANTIBIOTICS

1825

Escherichia coli 9.0 1

1~~

E

F

8.5

_

0 -j

8.01

7.5

0

5

10

15

20

25

20

25

TIME (hours)

B 9.5 9.0

D

, 8.5

0 -J

8.0.

7.5

0

5

10

15

TIME (hours) FIG. 1. (A) E. coli growth in three media. M9 alone (E), M9 with a minimal amount of a glucose carbon source (O), and M9 with an adequate glucose carbon source (V), representing media supporting no growth, slow growth, and optimal growth, respectively, (B) S. aureus in nongrowth, suboptimal-growth, and optimal-growth media represented by yeast nitrogen base alone (O), yeast nitrogen base with a glucose carbon source and additional amino acids (O), and yeast nitrogen base with a glucose carbon source and Casamino Acids (V), respectively.

at least 2 orders of magnitude more killing than a broadspectrum penicillin and an aminothiazolyl cephalosporin with inhibitory activity against these organisms. The differences in killing action among the antimicrobial agents were seen at 6 h, and these differences widened at 24 h of incubation. For slowly growing S. aureus, gentamicin and the two quinolones produced the best killing (nearly or greater than 3 orders of magnitude).

ANTIMICROB. AGENTS CHEMOTHER.

ENG ET AL.

1826

TABLE 1. Susceptibilities of test Organism and broth

organisms in

MIC

MERO IMIP CIP

(igWml)b

OFL GNT PIP NAF CTX RIF

E. coli

Mueller0.06 0.25 0.06 0.06 1.0 2.0 Hinton Suboptimal 0.015 0.12 0.015 0.015 1.0 0.25 0.06 0.25 0.06 0.06 1.0 2.0 Optimal S.

0.03 0.03

0.06

0.06 0.5

0.5

1

0.5

0.001

Suboptimal 0.03 0.03 Optimal

0.015 0.5

0.5 0.5

4 4

1.0 1.0

0.001 0.001

0.03 0.5

Log1o CFU/ml killed in the following phase:

Antibiotic (concn [I,g/ml])

Nongrowth

Suboptimal growth

Optimal growth

None (control) Meropenem (5) Imipenem (5) Ciprofloxacin (4) Ofloxacin (4) Gentamicin (8) Nafcillin (10) Rifampin (1)

0.9 1.1 1.0 1.5 1.4 0.9 1.0 0.7

-0.8 1.9 2.1 3.9 2.9 3.3 1.8 0.9

-1.1 3.8 3.1 4.5 4.4 1.9 3.5

0.03

aureus

MuellerHinton

TABLE 3. Summary of bactericidal activities against S. aureus at 24 ha

the media useda

Results are for an inoculum concentration of 5 x 10' CFU/ml. Abbreviations for antibiotics: MERO, meropenem; IMIP, imipenem; CIP, ciprofloxacin; OFL, ofloxacin; GNT, gentamicin; PIP, piperacillin; NAF, a

b

-0.2b

a Results are for a starting inoculum concentration of 108 CFU/ml. Negative numbers indicate growth. b Isolates resistant to rifampin developed at 24 h.

nafcillin; CTX, cefotaxime; RIF, rifampin.

Killing of rapidly growing bacteria. With rapidly growing bacteria, the quinolones, carbapenems, and gentamicin showed the greatest reduction in CFU for gram-negative bacteria. For S. marcescens, greater than 3-order-of-magnitude killing was observed with gentamicin at 6 h, but by 24 h regrowth occurred and total killing was reduced. Piperacillin produced the poorest bactericidal activity among TABLE 2. Summary of bactericidal activities against gram-negative bacteria at 24 ha Log1o CFU/ml killed

Medium

.

and antibiotic (concn

[>i.g/mlJ)

E-

K.

P.

E.

cloa-

S.

ccli

moniae

inosa

0.7 0.7

0.8 0.8 0.8 4.7 4.7 3.6 -0.1 -0.1

-0.1 -0.2 -0.1 1.8 1.8 -0.1 -0.1 -0.1

0.5 0.5 0.4 1.2 1.8 0.5

Nongrowth None (control) Meropenem (5) Imipenem (5) Ciprofloxacin (4) Ofloxacin (4) Gentamicin (8) Piperacillin (25) Cefotaxime (10)

5.7 4.7 1.4 -0.1 -0.1

0.4 0.2 0.4 3.7 1.8 0.7 -0.4 0.4

Suboptimal growth None (control) Meropenem (5) Imipenem (5) Ciprofloxacin (4) Ofloxacin (4) Gentamicin (8) Piperacillin (25) Cefotaxime (10)

-0.1 4.1 4.5 5.8 5.9 >7.0 -0.1 4.1

-0.2 5.6 3.9 5.7 4.8 7.0 0.4 2.8

-0.5 2.6 2.8 6.1 4.8 5.9 -0.1 0.1

-0.4 6.8 4.8 4.0 2.7 2.1 0.7 1.8

-1.2 0.4 0.3 2.1 3.6

Optimal growth None (control) Meropenem (5) Imipenem (5) Ciprofloxacin (4) Ofloxacin (4) Gentamicin (8) Piperacillin (25) Cefotaxime (10)

-1.8 >7.0 5.9 >7.0 5.8 >7.0 -0.1 5.9

-0.8 6.8 6.7 5.9 4.8 6.9 0.7 5.8

-1.2 3.8 1.8 4.9 4.8 6.9 -0.2 0.9

-1.1 6.7 5.4 2.9 2.7 5.9 -0.1 3.9

-1.3 1.9 0.8 -0.2 3.3 0.5b -0.1 -0.1

0.8

0.0 0.2

J.lb -0.1 -0.1

Data are based on a starting inoculum concentraiion of 10i CFU/ml. Negative numbers indicate growth. b For slowly or optimally growing S. marcescens, gentamicin produced greater than 3 orders of magnitude of killing at 6 h but regrowth occurred by a

24 h.

the antibiotics tested. These findings agree with previous observations on these antibiotics (3, 7). For S. aureus, the two quinolones tested appeared to have the best activity, which is consistent with previous observations (14). Gentamicin and rifampin appeared to kill optimally growing staphylococci initially, but by 24 h there was regrowth by resistant organisms. Optical density measurements. Initially, optical densities of the organisms were monitored along with viability determination of all of the test samples. In the nongrowing phase, we observed a significant decrease in turbidity without a concomitant decrease in viability in all of the gramnegative bacteria studied. This decrease was seen after the first 6 h and after 24 h of incubation. An example of this is shown in Fig. 2 for E. coli. After this observation, optical density was no longer used as a reliable measurement of bacterial killing. DISCUSSION Most antibiotics, especially the older P-lactams, kill bacteria only during the growth phase (15). During less-thanoptimal growth, a detailed study of a mutant auxotrophic E. coli strain showed that the carbapenem imipenem may offer a substantial measure of bactericidal activity during a 6-h observation (5). Bactericidal activity was monitored by optical density and bacterial viability. For longer observation periods, a chemostat was used to study antibiotic bactericidal activity for bacteria growing at different rates

(6).

Our observations extend the observations of Tuomanen et al. (5, 15) to nonauxotrophic gram-positive and gram-negative organisms, those that usually infect humans. Under conditions of no growth, we found that only the fluoroquinolones studied showed significant bactericidal activities. At 8 ,ug/ml, gentamicin showed no bactericidal activity. During slow growth, with the growth rate controlled by the amount of the carbon source added, several antibiotics shoWed substantial bactericidal activities. Among these were the two carbapenems studied, imipenem and meropenem. The clinical significance of the differences observed among the antibiotics for slowly growing or nongrowing bacteria is only speculative. It is known that there are many clinical situations in which the bacteria infecting the host are in a nearly dormant state, and this has recently been proven in an animal model (4). The slow growth or dormant state of

BACTERICIDAL EFFECTS OF ANTIBIOTICS

VOL. 35, 1991

9

120

7

90

0

6 IL

5 60

0 c

0

-J

° E 0

E :3

1827

co

4 0

3

30

a

0

I

0

0 11 12 TIME (hours) Escherichla coil

FIG. 2. Changes in optical density (OD) at 600 nm and bacterial viability observed over a 24-h period. Note the lack of correlation between optical density and viability. While both ofloxacin and meropenem caused similar reductions in optical density, only ofloxacin significantly reduced bacterial viability.

the bacteria may be responsible for bacteria escaping the antibiotic effect. In an animal-based surgical stitch model, the killing effects of antibiotics were dramatically reduced by the presence of a foreign body (suture threads) which can harbor organisms in a dormant state (1). S. epidermidis can produce large amounts of slirpe during the late log phase, which can further limit the'aivailability of nutrients to the organism and induce the organism to enter a state of dormancy (2, 9). Prosthetic implants are particularly susceptible to chronic colonization, since a protective environment may be available for the contaminating bacteria, thus permitting them to become dormant. Many infections, once established and addressed by a nearly normal host, become indolent and contain nonmultiplying bacteria. These can be encountered clinically in the form of abscesses or small pockets of walled-off infections (13). In all of these situations, antibiotics with substantial btctericidal activity for nonproliferating bacteria may have an advantage (16). The in vitro observations presented here need to be studied in an in vivo system to determine whether the differences observed among the antibiotics are clinically valid.

4.

5.

6.

7.

8. 9.

10. REFERENCES 1. Boon, R. J., and A. S. Beale. 1987. Response of Streptococcus pyogenes to therapy with amoxicillin or amoxicillin-clavulanic acid in a mouse model of mixed infection caused by Staphylococcus aureus and Streptococcus pyogenes. Antimicrob. Agents Chemother. 31:1204-1209. 2. Brown, M. R. W., D. G. Allison, and P. Gilbert. 1988. Resistance of bacterial biofilms to antibiotics: a growth related effect? J. Antimicrob. Chemother. 22:777-780. 3. Cherubin, C. E., and R. H. K. Eng. 1986. Experience with the

11.

12. 13.

use of cefotaxime in the treatment of bacterial meningitis. Am. J. Med. 80:398-404. Churd, C., J. C. Lucet, P. Rohner, M. Herrman, R. Auckenthaler, F. A. Waldvogel, and D. P. Lew. 1991. Resistance of Staphylococcus aureus recovered from infected foreign body in vivo to killing by antimicrobials. J. Infect. Dis. 163:1369-1373. Cozens, R. M., Z. Markiewicz, and E. Tuomanen. 1989. Role of autolysins in the activities of imipenem and CGP 31608, a novel penem, against slowly growing bacteria. Antimicrob. Agents Chemother. 33:1819-1821. Cozens, R. M., E. Tuomanen, W. Tosch, 0. Zak, J. Suter, and A. Tomasz. 1986. Evaluation of bactericidal activity of P-lactam antibiotics on slowly growing bacteria culturpd in the chemostat. Antimicrob. Agents Chemother. 29:797-802. Eng, R. H. K., C. E. Cherubin, J. C. Pechere, and T. R. Beam, Jr. 1987. Treatment failures of cefotaxime and lamatoxef in meningitis caused by Enterobacter and Serratia spp. J. Antimicrob. Chemother. 20:903-911. Eng, R. H. K., S. M. Smith, C. E. Cherubin, and E. N. Tan. 1991. Evaluation of two methods of overcoming the antibiotic carry-over effect. Eur. J. Microbiol. Infect. Dis. I:34-38. Evans, R. C., and C. J. Holmes. 1987. Effect of vancomycin hydrochloride on Staphylococcus epidermidis biofilm associated with silicone elastomer. Antimicrob. Agents Chemother. 31:889-894. National Committee for Clinical Laboratory Standards. 1987. Methods for determining bactericidal actiVity of antimicrobial agents. National Committee for Clinical Laboratory Standards, Villanova, Pa. National Committee for Clinical Laboratory Standards. 1990. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically-second ed. National Committee for Clinical Laboratory Standards, Villanova, Pa. Sambrook, J., E. F. Fritsch,'and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed., p. A.1-A.13. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Sheldon, H. 1988. Boyd's introduction to the study of disease,

1828

ENG ET AL.

10th ed., p. 129-155. Lea & Febiger, Philadelphia. 14. Smith, S. M., and R. H. K. Eng. 1985. Activity of ciprofloxacin for methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 27:688-691. 15. Tuomanen, E. 1986. Phenotypic tolerance: the search for 1-lactam antibiotics that kill nongrowing bacteria. Rev. Infect. Dis.

8(Suppl. 3):S279-S291.

ANTIMICROB. AGENTS CHEMOTHER. 16. Widmer, A. F., R. Frei, Z. Rajacic, and W. Zimmerli. 1990. Correlation between in vivo and in vitro efficacy of antimicrobial agents against foreign body infections. J. Infect. Dis. 162:96-102. 17. Wu, P. J., and D. M. Livermore. 1990. Response of chemostat cultures of Pseudomonas aeruginosa to carbapenems and other P-lactams. J. Antimicrob. Chemother. 25:891-902.