In vitro and in vivo activities of meropenem and comparable antimicrobial agents against Haemophilus influenzae, including ß-lactamase-negative ampicillin-resistant strains

Shuichi Miyazakia,*, Toshihiko Fujikawaa, Katsumori Kanazawab and Keizo Yamaguchia

a Department of Microbiology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo143-8540; b Sumitomo Pharmaceuticals Research Centre, Osaka, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro activity of ampicillin, cefotaxime, meropenem, panipenem, imipenem and biapenem was assayed using ampicillin-susceptible, ß-lactamase-positive and ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae isolated recently in Japan. Against ampicillin-susceptible isolates, cefotaxime was the most potent (MIC90 0.016 mg/mL). Both cefotaxime and meropenem (MIC90 of both, 0.5 mg/L) were the most potent against ß-lactamase-positive isolates. Against BLNAR isolates, meropenem (MIC90 0.5 mg/L) was the most potent. In murine bronchopneumonia caused by ampicillin-susceptible and BLNAR H. influenzae, cefotaxime showed the best efficacy, followed by meropenem. Our results indicate that meropenem could be a useful intravenous agent for infections caused by H. influenzae, including BLNAR strains.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Meropenem is a carbapenem with broad-spectrum potent activity against Gram-negative and Gram-positive bacteria. In particular, the in vitro activity of meropenem against Haemophilus influenzae is similar to that of third generation cephems.1

Of H. influenzae strains isolated during 1994–1995 in the USA, 36.4% were found to produce ß-lactamase, but the proportion of ß-lactamase-negative ampicillin-resistant (including intermediately susceptible) (BLNAR) isolates was 2.5%.2 In contrast, during 1996–1997, 14.9% of H. influenzae strains isolated in Japan were ß-lactamase positive while the proportion of BLNAR isolates was 44.4% of all ß-lactamase-negative isolates.3 The in vitro activity of cephems against BLNAR organisms is lower than that against ampicillin-susceptible organisms.3

We developed a murine model of bronchopneumonia induced by intranasal instillation of cell-bound H. influenzae.4 The purpose of the present study was to determine whether meropenem is effective in eliminating H. influenzae, especially BLNAR isolates in this model. The efficacies of various carbapenems (meropenem, imipenem, panipenem and biapenem), cefotaxime and ampicillin were evaluated by determination of viable bacterial counts in infected tissues.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

Thirty-one ß-lactamase-negative isolates, 34 ß-lactamase-positive isolates and 38 BLNAR isolates were studied. Ampicillin-susceptible strain TUM8 and BLNAR strain TUH267 were isolated from patients with bronchopneumonia admitted to our hospital.

Antimicrobial agents

The antibiotics evaluated were as follows: meropenem (Sumitomo Pharmaceuticals Co., Tokyo, Japan), imipenem/ cilastatin (Banyu Pharmaceutical Co., Tokyo, Japan), panipenem–betamipron (Sankyo Co., Tokyo, Japan), biapenem (Wyeth Lederle Japan, Tokyo, Japan), cefotaxime (Aventis Pharma, Tokyo, Japan) and ampicillin (Meiji Seika Co., Tokyo, Japan).

In vitro susceptibility test

MICs were determined using a microbroth dilution method. The medium used was cation-adjusted Mueller–Hinton broth supplemented with 5% lysed horse blood, 5 mg/mL yeast extract (Oxoid, Basingstoke, UK) and 15 µg/mL of NAD (Sigma Chemical Co., St Louis, MO, USA). The MICs were determined following incubation at 35°C for 18 h.

Experimental bronchopneumonia caused by H. influenzae

The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Toho University School of Medicine. The effects of various antimicrobial agents on bronchopneumonia caused by H. influenzae were examined using a mouse model described previously.4 Three days after treatment with formalin to induce airway injury, 50 µL of a cell-bound H. influenzae suspension (1.9–4.2 x 104 cfu/animal) was instilled intranasally into anaesthetized ICR mice (Nihon Charles River Co., Kanagawa, Japan). At 48 h after infection, viable counts in lower respiratory organs were 2–5 x 105 cfu/tissue. The test drug was administered to each group of mice (n = 8) at the dose recommended by the supplier. Specifically, the recommended dose of meropenem was 500 mg bd; imipenem–cilastatin, 500 mg (as imipenem) bd; panipenem– betamipron, 500 mg (as panipenem) bd; biapenem, 300 mg bd (personal communication); cefotaxime, 1000 mg bd and ampicillin, 500 mg qds. Based on this schedule, we elected to use each carbapenem at 20 mg/kg, and cefotaxime and ampicillin both at 40 mg/kg, injected sc. The frequency and duration of treatment of all test antimicrobials was twice a day for 3 days. Mice were killed 20 h after the last dose of the antimicrobial agent.

For determination of viable bacterial counts, the lungs and trachea were removed and homogenized in 2 mL of sterile saline, and 0.1 mL aliquots of serial 10-fold dilutions of the homogenate were plated on chocolate agar. Since the suspension of original tissue homogenate inhibited growth of H. influenzae, but a 10-fold dilution of it did not, the detectable limit of bacteria was 2 x 102 cfu/tissue.

Results are expressed as mean ± s.d. of log cfu/tissue. Differences between groups were examined for statistical significance using the Mann–Whitney U-test.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
MICs of meropenem and other antimicrobial agents for recent clinical isolates

The antibacterial activity (MIC90 0.125 mg/L) of meropenem against ampicillin-susceptible isolates was one-quarter that of cefotaxime and four or more times that of the other four antimicrobial agents (Table 1Go). Against ß-lactamasepositive and BLNAR isolates, the antibacterial activities (MIC90 for both, 0.5 mg/L) of meropenem were 25% of that for ampicillin-susceptible isolates. This activity was as good as that of cefotaxime and >=16 times that of other test drugs.


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Table 1. Antibacterial activity of meropenem and other parenteral antimicrobial agents against clinical isolates of H. influenzae
 
Efficacy of various antimicrobial agents in mice infected with H. influenzae

In the strain TUM8 infections, the number of bacteria in mice treated with cefotaxime decreased significantly, compared with those treated with other antimicrobials and controls (Table 2Go), and in six mice was less than the detection limit. Viable counts in mice treated with biapenem and ampicillin decreased significantly compared with the control, imipenem–cilastatin and panipenem–betamipron. Those in mice treated with meropenem–cilastatin decreased significantly, compared with the control and panipenem– betamipron. In mice treated with panipenem–betamipron and imipenem–cilastatin the number of bacteria was significantly lower than the control. Among meropenem– cilastatin-, biapenem- and ampicillin-treated mice, in one, one and two mice, respectively, viable counts in the infected tissues were less than the detection limit.


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Table 2. Efficacy of meropenem and comparable antimicrobial agents in murine bronchopneumonia caused by H. influenzae
 
In strain TUH267 infections, viable counts in infected tissues decreased in meropenem–cilastatin-, imipenem– cilastatin- and cefotaxime-treated mice, and were significantly lower than those of the control and mice treated with other test drugs.


    Discussion
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 Materials and methods
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The prevalence of ampicillin-resistant strains of H. influenzae has increased progressively in the UK, USA and Japan.2,3,5 There are two known resistance mechanisms; the first involves production of ß-lactamase while the second, which involves elaboration of altered penicillin-binding proteins (PBPs), is referred to as BLNAR.2 The latter have remained relatively uncommon in the USA and UK,2,5 whereas in Japan they occur more frequently.3 Since the MIC90 of cefotaxime for BLNAR isolates was 64 times higher than that for ampicillin-susceptible isolates in this study, there is a possibility that BLNAR organisms acquire cefotaxime resistance due to frequent administration of this drug.

NCCLS breakpoints of meropenem and imipenem for H. influenzae are <=0.5 and <=4 mg/L, respectively.6 In the present study, none of the isolates was resistant to meropenem, although we identified three ß-lactamase-positive isolates (0.9%) and 19 BLNAR isolates (50%) that were resistant to imipenem. Kanazawa et al.7 suggested that one of the reasons for this difference is that the introduction of the 1ß-methyl group or reduction of basicity of the C-2 side chain could increase the bactericidal activity of meropenem against H. influenzae due to increased affinity of the bacteria for PBP-4 and PBP-5.

Since meropenem is not stable against mouse DHP-I, similar to imipenem and panipenem,8 a mixture of meropenem and cilastatin (at a ratio of 1:1) was injected to prevent inactivation of meropenem. Meropenem–cilastatin, cefotaxime and imipenem–cilastatin were the most effective of the antimicrobials tested in the BLNAR infection model.

In conclusion, meropenem can be considered for the treatment of infections caused by imipenem-resistant H. influenzae and may also be useful for cefotaxime-resistant H. influenzae infections.


    Notes
 
* Corresponding author. Tel: +81-3-3762-4151; Fax: +81-3-5493-5415; E-mail: shuichi{at}med.toho-u.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Sumita, Y., Inoue, M. & Mitsuhashi, S. (1989). In vitro antibacterial activity and beta-lactamase stability of the new carbapenem SM-7338. European Journal of Clinical Microbiology and Infectious Diseases 10, 908–16.

2 . Doern, G. V., Brueggemann, A. B., Pierce, G., Holley, H. P. & Rauch, A. (1997). Antibiotic resistance among clinical isolates of Haemophilus influenzae in the United States in 1994 and 1995 and detection of ß-lactamase-positive strains resistant to ampicillin– clavulanate: Results of a national multicenter surveillance study. Antimicrobial Agents and Chemotherapy 41, 292–7.[Abstract]

3 . Seki, H., Kasahara, Y., Ohta, K., Saikawa, Y., Sumita, R., Yachie, A. et al. (1999). Increasing prevalence of ampicillin-resistant, non-beta-lactamase-producing strains of Haemophilus influenzae in children in Japan. Chemotherapy 45, 15–21.[ISI][Medline]

4 . Miyazaki, S., Nunoya, T., Matsumoto, T., Tateda, K. & Yamaguchi, K. (1997). New murine model of bronchopneumonia due to cell-bound Haemophilus influenzae. Journal of Infectious Diseases 175, 205–9.[ISI][Medline]

5 . Williams, J. D., Powell, M., Fah, Y. S., Seymour, A. & Yuan, M. (1992). In vitro susceptibility of Haemophilus influenzae to cefaclor, cefixime, cefetamet and loracarbef. European Journal of Clinical Microbiology and Infectious Diseases 11, 748–51.[ISI][Medline]

6 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.

7 . Kanazawa, K., Nouda, H. & Sunagawa, M. (1997). Structure– activity relationships of carbapenem compounds to anti-Haemophilus influenzae activity and affinity for penicillin-binding proteins. Effect of 1 beta-methyl group and C-2 side chain. Journal of Antibiotics (Tokyo) 50, 162–8.[ISI][Medline]

8 . Fukasawa, M., Sumita, Y., Harabe, E. T., Tanio, T., Nouda, H., Kohzuki, T. et al. (1992). Stability of meropenem and effect of 1 beta-methyl substitution on its stability in the presence of renal dehydropeptidase I. Antimicrobial Agents and Chemotherapy 36, 157–9.

Received 27 March 2001; returned 10 July 2001; revised 23 July 2001; accepted 15 August 2001