Influence of inoculum size of Staphylococcus aureus and Pseudomonas aeruginosa on in vitro activities and in vivo efficacy of fluoroquinolones and carbapenems

Shingo Mizunaga*, Tomoko Kamiyama, Yoshiko Fukuda, Masahiro Takahata and Junichi Mitsuyama

Research Laboratories, Toyama Chemical Co. Ltd, 2-4-1 Shimo-Okui, Toyama, Japan


* Corresponding author. Tel: +81-76-431-8268; Fax: +81-76-431-8208; E-mail: shingo_mizunaga{at}toyama-chemical.co.jp

Received 16 July 2004; returned 8 September 2004; revised 17 February 2005; accepted 20 April 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: We investigated the effect of inoculum size on MIC, bactericidal activity and the post-antibiotic effect (PAE) of carbapenems (imipenem, panipenem and meropenem) and injectable quinolones (pazufloxacin and ciprofloxacin) against Staphylococcus aureus and Pseudomonas aeruginosa, and also the relationship between in vivo systemic infection by changing the inoculum size injected in mice.

Results: Increasing the bacterial inoculum (105–108 cfu/mL) had no significant effect on the MIC of any of the tested antimicrobial agents. With the standard inocula (106 cfu/mL) of both test strains, all the antimicrobial agents showed bactericidal activity; however, increasing the inoculum size to >108 cfu/mL resulted in a reduction in bactericidal activity of all the antimicrobial agents against S. aureus Smith. In contrast, increasing the inoculum size of P. aeruginosa exerted only a minimal influence on the bactericidal activity of fluoroquinolones, but resulted in a reduction in the bactericidal activity of carbapenems. With the standard inoculum size of S. aureus Smith, pre-incubation with fluoroquinolones and carbapenems, except for meropenem, was sufficient to produce PAEs. When the inoculum was increased, the duration of the PAEs of these antimicrobial agents was reduced; however, those of fluoroquinolones were longer than carbapenems. Inoculum size had a greater influence on the in vivo efficacy of carbapenems than that of fluoroquinolones.

Conclusions: Our results suggest that decreased bactericidal activity, or the in vitro PAE of carbapenems and fluoroquinolones, is related to the reduced in vivo protective effect against infection caused by high inoculum with S. aureus or P. aeruginosa.

Keywords: bactericidal activity , post-antibiotic effect , protective effect


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Staphylococcus aureus and Pseudomonas aeruginosa are both important pathogens in severe clinical infections such as sepsis and nosocomial pneumonia, and are still associated with severe sequelae and mortality despite the use of appropriate antimicrobial agents.1,2 Therefore, it is crucially important that the initial choice of antimicrobial agent be correct if the treatment is to be successful. Injectable quinolones and carbapenems, both with broad antibacterial spectrum and strong antibacterial activity, have been used mainly for severe infections.35

Antibiotic susceptibility testing is a fundamental part of microbiological assessment and is supposed to aid the clinician in the choice of the most appropriate antibiotic therapy. The routine procedures of in vitro susceptibility testing are highly standardized and so may not reflect the in vivo situation of every patient and site of infection. Instead, the composition of media, culture condition, inoculum size and many other variables in in vitro tests may deviate significantly from the situation in patients. These discrepancies may have profound effects on antimicrobial activity. For example, hypoxic, anaerobic or acidic conditions prevailing in vivo during some infections may reduce the activity of several groups of antibiotics.68 Also, it was reported that inoculum size may alter antibiotic activity in vitro,911 and that bacterial concentrations in pus and in infected peritoneal fluid from patients were much higher than the inocula used in in vitro susceptibility tests.12

The effect of inoculum size on in vitro antibacterial activity is well studied;911 however, there are few reports discussing the effect of inoculum size on the in vivo efficacy of antimicrobial agents.13,14 Chuang et al.14 investigated the in vivo efficacy of antimicrobial therapy against severe thigh infection caused by Vibrio vulnificus, in experiments that used large inocula for infection.

In this study, we investigated the relationship between the effect of inoculum size on in vitro activities [MIC, bactericidal activity and post-antibiotic effect (PAE)] of carbapenems and injectable quinolones against S. aureus and P. aeruginosa, and how in vivo systemic infection could be modified by changing the inoculum size injected in mice.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

S. aureus Smith and the clinical isolate P. aeruginosa S-1294 were used in this study. These organisms were stored at –130°C in skimmed milk (Difco Laboratories, Detroit, MI, USA) until use.

Media and chemicals

Mueller–Hinton agar (MHA), Mueller–Hinton broth (MHB) and mucin bacteriological were purchased from Difco Laboratories (Detroit, MI, USA). The following agents were used in this study. Pazufloxacin mesilate was synthesized at Toyama Chemical Co., Ltd (Tokyo, Japan). Ciprofloxacin was extracted from commercially available tablets, purchased from Bayer Yakuhin Co., Ltd (Osaka, Japan). The purity of each of these two agents was >99.8%, as measured by high-performance liquid chromatography. Imipenem–cilastatin, panipenem–betamipron and meropenem were purchased from Banyu Pharmaceutical Co., Ltd (Tokyo, Japan), Sankyo Co., Ltd (Tokyo, Japan) and Sumitomo Pharmaceuticals Co., Ltd (Tokyo, Japan), respectively. The MICs and ED50s of pazufloxacin mesilate, imipenem–cilastatin and panipenem–betamipron were equivalent to those of pazufloxacin, imipenem and panipenem, respectively.

Animals

The 3.5-week-old male ICR strain mice (body weight 15 to 17 g) were purchased from Japan SLC, Inc. (Shizuoka, Japan) and assigned to this study after an acclimatization period of 3 days. On the day of infection, the mice were randomly allocated into groups of 10. All animal experimental procedures were conducted in accordance with the guidelines for care and use of laboratory animals at Toyama Chemical Co., Ltd.

In vitro study

Susceptibility testing. MICs were determined by the standard agar dilution method according to the guidelines of the Japan Society of Chemotherapy.15 The bacteria were tested at various inocula size (105–108 cfu/mL) by using a multipoint inoculator (Sakuma Seisakusyo, Tokyo, Japan), and were incubated in ambient air at 37°C for 24 h. The lowest drug concentration showing no growth was determined as the MIC.

Bactericidal activity. Bactericidal activity was determined by the time–kill curve method. Test tubes containing 60 µL of the appropriate antimicrobial agent were inoculated with 6 mL of each test organism in cation-adjusted MHB, which was pre-incubated at 37°C for 1 h (adjusted to a density of ~106 or 108 cfu/mL) to yield a drug concentration equivalent to 2x, 8x or 16 x MIC. Initial inocula sizes of ~106 and 108 cfu/mL for each strain were used as standard and higher, respectively. The test tubes were incubated at 37°C in a water bath. Aliquots were removed at 0, 0.5, 1, 2, 4, 6, 8 and 12 h, serially diluted in sterilized saline solution, and plated on an MHA plate to determine the viable cell count. Total bacterial counts (cfu/mL) were determined after 24 h of incubation at 37°C. The detection limit of colony counts was 25 cfu/mL. The antimicrobial agents were considered bactericidal when they reduced the original inoculum by ≥3 log10 cfu/mL (99.9%) at each of the time points, and the antimicrobial agents were considered bacteriostatic when the inoculum was reduced by 0 to 3 log10 cfu/mL.

In vitro PAE. The inoculum with 6 mL of each test organism, which was pre-incubated at 37°C for 1 h (adjusted to a density of ~106 or 108 cfu/mL), was exposed to antimicrobial agents at 2x or 16 x MIC for 1 h (S. aureus Smith) and 15 min (P. aeruginosa S-1294). As with determination of bactericidal activity, inoculum sizes of ~106 and 108 cfu/mL for each strain were used as standard and higher, respectively.

After incubation with each agent, the bacteria were washed three times with sterilized physiological saline on a membrane filter (pore size 0.2 µm, millipore filter, Schleicher & Schuell, Germany) and transferred to drug-free fresh medium. In the case of 108 cfu/mL, the inoculum was diluted with drug-free fresh medium (1:1000) before washing the bacteria, because of the difficulty of filtration. The resulting re-growth curve was constructed by determination of viable cell count at the time of drug removal and at appropriate intervals (2 h) thereafter.

The duration of PAE was calculated by means of the equation PAE = T – C, where T is the time required for the viable count in the test culture to increase 1 log10 above the count immediately after the dilution procedure, and C is the time required for the cfu count in an untreated culture to increase by 1 log10 above the count observed after drug removal.

In vivo study

In vivo activity was determined for mice with systemic infection of varying inoculum size. The bacterial cells, which were prepared from an overnight culture on MHA at 37°C, were suspended in sterilized saline solution. The inocula were obtained by 10-fold dilution in 1/15 M phosphate buffer containing 5.6% mucin. Mice were inoculated intraperitoneally with 0.5 mL of bacterial suspension corresponding to different inocula. According to the results of a preliminary study, 1 x 107 and 1 x 109 cfu/mouse for S. aureus Smith, and 4 x 105 and 4 x 107 cfu/mouse for P. aeruginosa S-1294 were used as inocula to represent the infection caused by a low or high inoculum, respectively. With these inocula, all the mice died within 3 days. A few mice infected with the 106 cfu/mouse for S. aureus Smith or 104 cfu/mouse for P. aeruginosa S-1294 survived for >7 days.

Pazufloxacin mesilate, imipenem–cilastatin, panipenem–betamipron and meropenem were dissolved and diluted in sterilized saline solution. Ciprofloxacin was dissolved in 0.1 M NaOH and diluted in sterilized saline solution. The diluted agents were administered subcutaneously at 1 h after infection. Six to nine dose levels (1.4-fold serial dilution) were tested for each antimicrobial agent. The total number of surviving mice at day 7 post-infection was recorded, and then the 50% effective dose (ED50) and 95% confidence limits were determined by the Probit method.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro study

We investigated the effect of inoculum size on the MICs of the antimicrobial agents against S. aureus Smith and P. aeruginosa S-1294. At the standard inoculum size (105 cfu/mL), the MICs of pazufloxacin, ciprofloxacin, imipenem, panipenem and meropenem were 0.2, 0.2, 0.025, 0.025 and 0.1 mg/L for S. aureus Smith, and 0.39, 0.2, 1.56, 6.25 and 0.39 mg/L for P. aeruginosa S-1294, respectively. Increasing the bacterial inoculum (105 to 108 cfu/mL) had no significant effect (more than four-fold) on the MIC of any of the tested antimicrobial agents (data not shown).

The results of the time–kill studies are presented in Figures 1 and 2.



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Figure 1. Time–kill studies of pazufloxacin, ciprofloxacin, imipenem, panipenem and meropenem against S. aureus Smith using inocula of 1 x 106 and 1 x 108 cfu/mL. Continuous lines, 1 x 106 cfu/mL; broken lines, 1 x 108 cfu/mL.

 


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Figure 2. Time–kill studies of pazufloxacin, ciprofloxacin, imipenem, panipenem and meropenen against P. aeuruginosa S-1294 using inocula of 2 x 106 and 2 x 108 cfu/mL. Continuous lines 2 x 106 cfu/mL; broken lines 2 x 108 cfu/mL.

 
For S. aureus Smith (Figure 1) with the standard inoculum (1 x 106 cfu/mL), pazufloxacin, imipenem and panipenem achieved 99.9% killing after 4 h, at a concentration of 2 x MIC, and ciprofloxacin and meropenem achieved 99.9% killing after 6 h, at the same multiple of MIC. All agents achieved 99.9% killing after 2 h, at the concentration of 16 x MIC. When the inoculum was increased to 1 x 108 cfu/mL, none of the antimicrobial agents showed bactericidal activity at concentrations of 2–16 x MIC.

Pazufloxacin and ciprofloxacin achieved 99.9% killing against P. aeruginosa S-1294 (Figure 2) with the standard inoculum (2 x 106 cfu/mL) at 8x and 16 x MIC after 0.5 h. Imipenem, panipenem and meropenem showed the same results at 16 x MIC after 4 h. When the inoculum was increased to 2 x 108 cfu/mL, imipenem, panipenem and meropenem did not achieve >99.9% killing at 2–16 x MIC. In contrast, pazufloxacin and ciprofloxacin achieved 99.9% killing, at 8x or 16 x MIC after 1 h on increasing the inoculum size of P. aeruginosa.

The data from the in vitro PAE experiments are shown in Table 1.


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Table 1. Effect of inoculum size on in vitro PAE of pazufloxacin, ciprofloxacin, imipenem, panipenem and meropenem against S. aureus Smith and P. aeruginosa S-1294

 
With the standard inoculum size (5 x 106 cfu/mL) of S. aureus Smith, pre-incubation with pazufloxacin, ciprofloxacin, imipenem and panipenem at a concentration of 16 x MIC was sufficient to produce PAEs of 2.2, 2.0, 2.5 and 1.9 h, respectively. Meropenem had minimal effect (0.6 h) with even the standard inoculum size. When the inoculum was increased to 7 x 108 cfu/mL, the duration of the PAEs of these antimicrobial agents was reduced; however, those of pazufloxacin and ciprofloxacin at 16 x MIC (1.3 and 1.0 h, respectively) were longer than those of imipenem and panipenem (0.5 and 0.5 h, respectively).

With the standard inoculum size (7 x 106 cfu/mL) of P. aeruginosa S-1294, pre-incubation with pazufloxacin, ciprofloxacin and meropenem at a concentration of 16 x MIC was sufficient to produce PAEs of 1.9, 2.0 and 1.2 h, respectively. Imipenem and panipenem had minimal effect (0.4 and 0.3 h, respectively) with even the standard inoculum size at the same multiple of MIC. When the inoculum was increased to 8 x 108 cfu/mL, the duration of the PAEs of pazufloxacin, ciprofloxacin and meropenem were reduced; however, those of pazufloxacin and ciprofloxacin at 16 x MIC (1.1 and 1.0 h, respectively) were longer than that of meropenem (0.5 h). With the higher inocula of S. aureus Smith and P. aeruginosa S-1294, the duration of the PAE of fluoroquinolones was longer than that of carbapenems.

In vivo efficacy

As shown in Table 2, the protective efficacy of antimicrobial agents was compared by changing the inoculum size of S. aureus Smith and P. aeruginosa S-1294 administered to mice.


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Table 2. Protective effect of pazufloxacin, ciprofloxacin, imipenem, panipenem and meropenem on experimental systemic infection by changing the inoculum size

 
Against S. aureus Smith, the ED50s of pazufloxacin and ciprofloxacin were ~20–28x higher for the infection caused by the high inoculum (1 x 109 cfu/mouse) than for the infection caused by the low inoculum (1 x 107 cfu/mouse). The ED50s of imipenem and panipenem were about 900–1000 times higher for the infection caused by the high inoculum than for the infection caused by the low inoculum. Meropenem was therapeutically ineffective at the dose tested against the infection caused by the high inoculum.

Against P. aeruginosa S-1294, the ED50s of pazufloxacin and ciprofloxacin were about 11–23 times higher for the infection caused by the high inoculum (4 x 107 cfu/mouse) than for the infection caused by the low inoculum (4 x 105 cfu/mouse). On the other hand, imipenem, panipenem and meropenem were therapeutically ineffective at the dose tested against the infection caused by the high inoculum.

For S. aureus Smith and P. aeruginosa S-1294, inoculum size had a larger influence on the efficacy of carbapenems than that of fluoroquinolones.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MIC is the main microbiological parameter used to predict the efficacies of antibiotics. However, it is well known that MICs may vary according to the inoculum size used (inoculum effect), especially with some ß-lactam antibiotics.16,17 In our study, the MICs of all tested antimicrobial agents were not affected by an increase in inoculum size. In contrast, with in vivo activity, the inoculum size of S. aureus Smith and P. aeruginosa S-1294 influenced the protective efficacy of carbapenems more than that of fluoroquinolones, suggesting that there is no relationship between the MIC and the efficacy of antimicrobial agents for the infection caused by the high inoculum.

The time–kill technique is a standard method used to follow the kinetics of bacterial killing in vitro. All antimicrobial agents in this experiment showed bactericidal activity with the standard inocula of both test strains. However, increasing the inoculum size of S. aureus Smith from 1 x 106 to 1 x 108 cfu/mL resulted in a reduction in bactericidal activity of all antimicrobial agents. It was found that the decreased bactericidal activity was accompanied by reduced protective effects. However, the in vivo efficacy of fluoroquinolones was little affected by the inoculum size; compared with carbapenems the degree of the decreased bactericidal activity is about the same. In contrast, increasing the inoculum size of P. aeruginosa from 2 x 106 to 2 x 108 cfu/mL exerted only a minimal influence on the bactericidal activity of fluoroquinolones, but resulted in a reduction in the bactericidal activity of carbapenems. Significantly, the in vivo results paralleled the in vitro data; that is, the protective effects of fluoroquinolones were little affected by changes of inoculum size for infection. The ED50s of ß-lactam antibiotics in mice infected with P. aeruginosa have been reported as being markedly correlated with the ability of the drug to kill the bacteria in a short time (killing activity).18 This result correlates well with our observation; the protective effects of carbapenems were dramatically affected (therapeutically ineffective) by inoculum size, as well as by the lowering of the in vitro bactericidal activities. It is speculated that the difference between bactericidal activity of fluoroquinolones and that of carbapenems with the higher inoculum of P. aeruginosa S-1294 related to the in vivo results, i.e. inoculum size had a greater influence on the efficacy of carbapenems than that of fluoroquinolones.

The PAE, the phenomenon of continued bacterial growth inhibition after exposure to an antimicrobial agent, has become of clinical interest because the presence of a PAE may be an important consideration in designing antibiotic dosage regimens.19 It is unclear whether or not PAE is related to the in vivo protective effect; however, it has been suggested that an antimicrobial agent, which has a PAE, may modify the cell surface, thereby enhancing bacterial susceptibility to phagocytosis, intracellular killing or both in non-neutropenic animals.19,20 We investigated the relationship between the effects of inoculum size on the in vitro PAE of carbapenems and fluoroquinolones, and those of the protective effect against the infection caused by the high inoculum. The duration of the PAEs of the antimicrobial agents at 16 x MIC for the standard inoculum of S. aureus Smith, except for meropenem, were >1.9 h and the duration of the PAE of imipenem was the longest. On the other hand, with the higher inoculum of S. aureus Smith, the duration of the PAE of fluoroquinolones was longer than that of carbapenems. On consideration of the results of the bactericidal activity, it was speculated that the in vivo protective effect of fluoroquinolones for the infection caused by the high inoculum with S. aureus Smith was related to PAE rather than to bactericidal activity. Against P. aeruginosa S-1294, imipenem and panipenem had short PAEs even with the standard inoculum size. ß-Lactam antibiotics consistently induce a PAE against Gram-positive cocci, but there is no effect or only a minimal effect against Gram-negative bacilli. Carbapenems are unique among ß-lactams because they can induce significant in vitro PAE against P. aeruginosa;21,22 however, in our experiment, imipenem and panipenem had short PAEs against P. aeruginosa S-1294. Several factors that affect the PAE must be kept in mind: the drug–pathogen combination, duration of exposure, drug concentration, type of medium, mechanical agitation and growth of the pathogen.23,24 The duration of the PAE of imipenem for P. aeruginosa S-1294 was <0.5 h despite exposing the organism to 16 x MIC for 2 h (data not shown).

In this study, the in vivo protective effect of fluoroquinolones and carbapenems was investigated in animals by changing the inoculum size. For S. aureus and P. aeruginosa, inoculum size had a greater influence on the efficacy of carbapenems than that of fluoroquinolones. The results obtained in some animal species cannot be directly related to those in humans because biotransformation varies with the animal species. However, it may be possible to determine a trend from the results in the same animal species under definite conditions, and the results may give a clue to estimating the efficacy of antimicrobial agents in human infection. The clinical significance of the in vitro assessment of antibiotic activity is limited because both bacterial concentrations at the site of infection may differ significantly from the conditions used in standard susceptibility tests. Even if the strain is susceptible, other criteria have to be considered because of the importance of bacterial concentrations at the site of infection.

In conclusion, in vitro antimicrobial activity (bactericidal activity or PAE) and the in vivo efficacy of fluoroquinolones were little affected by the inoculum size, compared with carbapenems, suggesting that decreased bactericidal activity or the in vitro PAE of carbapenems and fluoroquinolones is related to the reduced in vivo protective effect against infection caused by the high inoculum with S. aureus or P. aeruginosa.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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