Microbiology Chemotherapy 1992;38:28-35
Department of Microbiology, Yamanashi Medical College, Yamanashi; Product Development Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka; Product Planning Department, Fujisawa Pharmaceutical Co., Ltd., Tokyo, Japan
Key Words ß-Lactamase Disk susceptibility Moraxella (Branhamella) catarrhalis Streptococcus pneumoniae Streptococcus pyogenes Haemophilus influenzae
Antibacterial Activity of Cefixime against and in the Presence of
Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae Moraxella (Branhamella) catarrhalis
Abstract We measured the sizes of the inhibition zones of oral P-lactam antibiotics for Streptococcus pneumoniae, Streptococcus pyo genes and Haemophilus influenzae in the presence of (Llactamase-producing-MoraYe//a (Branhamella) catarrhalis by the agar double-layer method. The sizes of the zones of amoxicillin for S. pneumoniae alone were the largest, followed in a de scending order by those of cefixime and cefaclor. In the pres ence of 107 CFU/ml of M.(B.) catarrhalis, however, significant reduction of the sizes of the zones was seen with amoxicillin and cefaclor; inhibition with cefixime was nearly unchanged. Similar results were observed in those for S. pyogenes. These variable findings were attributed to the difference in stability of these drugs to the p-lactamase produced by M.(B.) catarrha lis. When the susceptibility of H. influenzae in the presence of 108 CFU/ml of M.(B.) catarrhalis to cefixime, cefoteram, cefpodoxime, cefotiam and cefuroxime was examined, the sizes of the inhibition zones of all the drugs were reduced by the pres ence of 108 CFU/ml of M.(B.) catarrhalis, but those of cefixime were the largest of all the drugs tested. Our agar double-layer method is simple and useful for evaluating the influence of P-lactamase-producing organism, a s M.(B.) catarrhalis, on the disk susceptibility of other pathogens to antibiotics.
Yokota Yoshiko, PhD Dept, of Chemotherapy, Product Devel opment Laboratories Fujisawa Pharmaceutical Co., Ltd. 1-6,2-Chomc, Kashima, Yodogawa-Ku, Osaka (Japan)
© 1992
S. Karger AG, Basel 0009- 3157/92 / 0381-0028 $ 2.75/0
Downloaded by: Kings's College London 137.73.144.138 - 11/12/2017 2:05:31 PM
Toshihiko Yamadaa Yoshiko Yokotab Fumiaki Ikedab Yasuhiro Mineb Takahide Kitadac
p-Lactamase-producing Moraxella (Branhamella) catarrhalis now account for about 80% of the clinical isolates of the organism [1, 2]. Although in some cases it is the sole in fecting organism [3-6], it more often coexists with Haemophilus influenzae or Streptococcus pneumoniae [7, 8]. An important feature of mixed infection is the ability of organisms re sistant to an antimicrobial agent to protect others susceptible to that agent through pro duction of an antibiotic-degrading enzyme [9]. Clinical observations suggest that P-lactamase produced by M.(B.) catarrhalis might have the same importance in mixed infections [10- 12]. In this study, we investigated the influence of P-lactamase-producing- M.(B.) catarrhalis on the susceptibility of S. pneumoniae, S. pyo genes and H. influenzae to some (l-lactam anti biotics.
Materials and Methods Bacterial Strains S. pneumoniae, S. pyogenes, H. influenzae and M.(B.) catarrhalis isolated from clinical specimens from 1987 to 1990 were used. Antimicrobial Agents The following antibiotics were used: amoxicillin and cefixime (Fujisawa Pharmaceutical Co., Ltd., Japan), cefaclor (Sionogi Pharmaceutical Co., Ltd., Japan), ccfotiam (Takeda Pharmaceutical Co., Ltd., Japan), cefuroxime (Glaxo Japan Co., I .td., Japan) cefteram and cefpodoxime (synthesized in Fujisawa New Drug Research Laboratories, Japan). MICs by Agar Dilution Method MICs were determined by the agar dilution method specified by the Japan Society of Chemotherapy [13]. M.(B.) catarrhalis was incubated in Trypticase Soy broth (TSB, BBL) for 18 h and thereafter its 100-fold dilution was inoculated on Mueller Hinton agar (MHA; Difco) containing a serial twofold dilution of each drug with a multiplate inoculator. These plates were incubated at 37 °C for 18 h.
Disk Susceptibility Test by the Agar Double-Layer Method 30 ug/disk of each drug was used. M.(B.) catarrhalis No. 6017 ([1-lactamase-producing clinical isolate) was inoculated into MHA to make 107CFU/ml, and 5 ml of the MHA with M. (B.) catarrhalis was poured into petri dishes, solidified, and used as the lower layer. A further 5 ml of MHA containing 5% horse defibrinated blood was added to the dishes as upper layer to prepare the agar double-layer and solidified. 0.1 ml of 5. pneumo niae or S. pyogenes suspension adjusted to McFaland 0.5 (about 108 CFU/ml) was inoculated on the surface of the double-layer dishes and diffused with a Conradi bar. As a control, MHA with S. pneumoniae and with out M.(B.) catarrhalis (lower layer) was used. In the disk susceptibility test of H. influenzae, M.(B.) catarrha lis No. 6017 was inoculated into MHA to make 108 CFU/ml and used as the lower layer, and Brain Heart Infusion agar (BH1A; Difco) containing 10 pg/ml of hemin (Sigma Chemical Company, USA) and 10 pg/ml of |5-nicotinamide adenine dinucleotide (NAD; Sigma) was used as the upper layer medium. 0.1 ml of H. influ enzae suspension adjusted to McFaland 0.5 (about 108 CFU/ml) was inoculated on the surface of the doublelayer dishes and diffused with a Conradi bar. The size of the zone of inhibition was measured after incubation at 37 °C for 18 h. Preparation and Characterization o f ¡1-Lactamase of M.(B.) catarrhalis M.(B.) catarrhalis was incubated overnight on the MHA plate containing 1 pg/ml of amoxicillin, sus pended in the M/15 phosphate buffer solution (pH 7.0) and centrifuged at 8,000 r.p.m. for 10 min. After centrifugation, the pellet was resuspended in 1.5 ml of phosphate buffer solution, sonicated at 4°C by using an Insonator Model 200M (strength: 6, Ku bota, Japan) and centrifuged at 10,000 r.p.m. for 20 min. The supernatant was used as crude enzyme. The concentration of the protein was measured by Brad ford’s [14] method using bovine serum albumin as the standard solution. 50 pi of crude enzyme was added to 3 ml of the test drugs (final concentration: 50 pg/ml) and the absorption was measured with a Spectropho tometer (Type 220A, Hitachi, Japan) and the relative value was expressed as 100 for the rate of hydrolysis of amoxicillin (50 pg/ml). Bactericidal Activity S. pneumoniae No. 6001 (7 x 105 CFU/ml) and M.(B.) catarrhalis No. 6017 (1 x 107CFU/ml) were used. MIC of amoxicillin, cefaclor and cefixime against S. pneumoniae No. 6001 was 0.025 pg/ml, 0.2 pg/ml and
29
Downloaded by: Kings's College London 137.73.144.138 - 11/12/2017 2:05:31 PM
Introduction
a
b
Fig. 1. Photograph of disk sus ceptibility of S. pneumoniae in the presence of ß-lactamase-producing-M.(fl) catarrhalis by the agar double-layer method. A = Cefix ime; B = cefaclor; C = amoxicillin, a S. pneumoniae No. 6001 alone; b 5. pneumoniae No. 6001 in the pres ence of M. (B.) catarrhalis No. 6017.
Table 1. Disk inhibition zones of amoxicillin, cefaclor and cefixime against S. pneumoniae and S. pyogenes in the presence of M. (B.) catarrhalis No. 6017 by the agar double-layer method
Organism
5. pneumoniae (n = 20) S. pyogenes (n = 20)
M. (B.) catarrhalis1'
-
+ —
+
Mean inhibition zone diameter, mm amoxicillin
cefaclor
cefixime
37.0 ±2.03 13.6±3.97*b
29.5 ±2.00 11.0±3.70*c
28.2 ±2.77 27.7 ±2.68
35.0 ±1.69 11.5 ± 2.66* d
31.0 ±1.69 9.3 ± 1.46*-c
27.0 ±1.97 23.9 ±2.01*
n = Number of strains. * p
Department of Microbiology, Yamanashi Medical College, Yamanashi; Product Development Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka; Product Planning Department, Fujisawa Pharmaceutical Co., Ltd., Tokyo, Japan
Key Words ß-Lactamase Disk susceptibility Moraxella (Branhamella) catarrhalis Streptococcus pneumoniae Streptococcus pyogenes Haemophilus influenzae
Antibacterial Activity of Cefixime against and in the Presence of
Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae Moraxella (Branhamella) catarrhalis
Abstract We measured the sizes of the inhibition zones of oral P-lactam antibiotics for Streptococcus pneumoniae, Streptococcus pyo genes and Haemophilus influenzae in the presence of (Llactamase-producing-MoraYe//a (Branhamella) catarrhalis by the agar double-layer method. The sizes of the zones of amoxicillin for S. pneumoniae alone were the largest, followed in a de scending order by those of cefixime and cefaclor. In the pres ence of 107 CFU/ml of M.(B.) catarrhalis, however, significant reduction of the sizes of the zones was seen with amoxicillin and cefaclor; inhibition with cefixime was nearly unchanged. Similar results were observed in those for S. pyogenes. These variable findings were attributed to the difference in stability of these drugs to the p-lactamase produced by M.(B.) catarrha lis. When the susceptibility of H. influenzae in the presence of 108 CFU/ml of M.(B.) catarrhalis to cefixime, cefoteram, cefpodoxime, cefotiam and cefuroxime was examined, the sizes of the inhibition zones of all the drugs were reduced by the pres ence of 108 CFU/ml of M.(B.) catarrhalis, but those of cefixime were the largest of all the drugs tested. Our agar double-layer method is simple and useful for evaluating the influence of P-lactamase-producing organism, a s M.(B.) catarrhalis, on the disk susceptibility of other pathogens to antibiotics.
Yokota Yoshiko, PhD Dept, of Chemotherapy, Product Devel opment Laboratories Fujisawa Pharmaceutical Co., Ltd. 1-6,2-Chomc, Kashima, Yodogawa-Ku, Osaka (Japan)
© 1992
S. Karger AG, Basel 0009- 3157/92 / 0381-0028 $ 2.75/0
Downloaded by: Kings's College London 137.73.144.138 - 11/12/2017 2:05:31 PM
Toshihiko Yamadaa Yoshiko Yokotab Fumiaki Ikedab Yasuhiro Mineb Takahide Kitadac
p-Lactamase-producing Moraxella (Branhamella) catarrhalis now account for about 80% of the clinical isolates of the organism [1, 2]. Although in some cases it is the sole in fecting organism [3-6], it more often coexists with Haemophilus influenzae or Streptococcus pneumoniae [7, 8]. An important feature of mixed infection is the ability of organisms re sistant to an antimicrobial agent to protect others susceptible to that agent through pro duction of an antibiotic-degrading enzyme [9]. Clinical observations suggest that P-lactamase produced by M.(B.) catarrhalis might have the same importance in mixed infections [10- 12]. In this study, we investigated the influence of P-lactamase-producing- M.(B.) catarrhalis on the susceptibility of S. pneumoniae, S. pyo genes and H. influenzae to some (l-lactam anti biotics.
Materials and Methods Bacterial Strains S. pneumoniae, S. pyogenes, H. influenzae and M.(B.) catarrhalis isolated from clinical specimens from 1987 to 1990 were used. Antimicrobial Agents The following antibiotics were used: amoxicillin and cefixime (Fujisawa Pharmaceutical Co., Ltd., Japan), cefaclor (Sionogi Pharmaceutical Co., Ltd., Japan), ccfotiam (Takeda Pharmaceutical Co., Ltd., Japan), cefuroxime (Glaxo Japan Co., I .td., Japan) cefteram and cefpodoxime (synthesized in Fujisawa New Drug Research Laboratories, Japan). MICs by Agar Dilution Method MICs were determined by the agar dilution method specified by the Japan Society of Chemotherapy [13]. M.(B.) catarrhalis was incubated in Trypticase Soy broth (TSB, BBL) for 18 h and thereafter its 100-fold dilution was inoculated on Mueller Hinton agar (MHA; Difco) containing a serial twofold dilution of each drug with a multiplate inoculator. These plates were incubated at 37 °C for 18 h.
Disk Susceptibility Test by the Agar Double-Layer Method 30 ug/disk of each drug was used. M.(B.) catarrhalis No. 6017 ([1-lactamase-producing clinical isolate) was inoculated into MHA to make 107CFU/ml, and 5 ml of the MHA with M. (B.) catarrhalis was poured into petri dishes, solidified, and used as the lower layer. A further 5 ml of MHA containing 5% horse defibrinated blood was added to the dishes as upper layer to prepare the agar double-layer and solidified. 0.1 ml of 5. pneumo niae or S. pyogenes suspension adjusted to McFaland 0.5 (about 108 CFU/ml) was inoculated on the surface of the double-layer dishes and diffused with a Conradi bar. As a control, MHA with S. pneumoniae and with out M.(B.) catarrhalis (lower layer) was used. In the disk susceptibility test of H. influenzae, M.(B.) catarrha lis No. 6017 was inoculated into MHA to make 108 CFU/ml and used as the lower layer, and Brain Heart Infusion agar (BH1A; Difco) containing 10 pg/ml of hemin (Sigma Chemical Company, USA) and 10 pg/ml of |5-nicotinamide adenine dinucleotide (NAD; Sigma) was used as the upper layer medium. 0.1 ml of H. influ enzae suspension adjusted to McFaland 0.5 (about 108 CFU/ml) was inoculated on the surface of the doublelayer dishes and diffused with a Conradi bar. The size of the zone of inhibition was measured after incubation at 37 °C for 18 h. Preparation and Characterization o f ¡1-Lactamase of M.(B.) catarrhalis M.(B.) catarrhalis was incubated overnight on the MHA plate containing 1 pg/ml of amoxicillin, sus pended in the M/15 phosphate buffer solution (pH 7.0) and centrifuged at 8,000 r.p.m. for 10 min. After centrifugation, the pellet was resuspended in 1.5 ml of phosphate buffer solution, sonicated at 4°C by using an Insonator Model 200M (strength: 6, Ku bota, Japan) and centrifuged at 10,000 r.p.m. for 20 min. The supernatant was used as crude enzyme. The concentration of the protein was measured by Brad ford’s [14] method using bovine serum albumin as the standard solution. 50 pi of crude enzyme was added to 3 ml of the test drugs (final concentration: 50 pg/ml) and the absorption was measured with a Spectropho tometer (Type 220A, Hitachi, Japan) and the relative value was expressed as 100 for the rate of hydrolysis of amoxicillin (50 pg/ml). Bactericidal Activity S. pneumoniae No. 6001 (7 x 105 CFU/ml) and M.(B.) catarrhalis No. 6017 (1 x 107CFU/ml) were used. MIC of amoxicillin, cefaclor and cefixime against S. pneumoniae No. 6001 was 0.025 pg/ml, 0.2 pg/ml and
29
Downloaded by: Kings's College London 137.73.144.138 - 11/12/2017 2:05:31 PM
Introduction
a
b
Fig. 1. Photograph of disk sus ceptibility of S. pneumoniae in the presence of ß-lactamase-producing-M.(fl) catarrhalis by the agar double-layer method. A = Cefix ime; B = cefaclor; C = amoxicillin, a S. pneumoniae No. 6001 alone; b 5. pneumoniae No. 6001 in the pres ence of M. (B.) catarrhalis No. 6017.
Table 1. Disk inhibition zones of amoxicillin, cefaclor and cefixime against S. pneumoniae and S. pyogenes in the presence of M. (B.) catarrhalis No. 6017 by the agar double-layer method
Organism
5. pneumoniae (n = 20) S. pyogenes (n = 20)
M. (B.) catarrhalis1'
-
+ —
+
Mean inhibition zone diameter, mm amoxicillin
cefaclor
cefixime
37.0 ±2.03 13.6±3.97*b
29.5 ±2.00 11.0±3.70*c
28.2 ±2.77 27.7 ±2.68
35.0 ±1.69 11.5 ± 2.66* d
31.0 ±1.69 9.3 ± 1.46*-c
27.0 ±1.97 23.9 ±2.01*
n = Number of strains. * p
