Microbiology and Immunology - December 1991 Ouchi - Antibacterial Activity of Cefpodoxime Against Branhamella

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Microbiol. Immunol.
Vol. 35 (12), 1059-1071, 1991
Antibacterial Activity of Cefpodoxime against

Branhamella catarrhalis
Takashi TAKENOUCHI, * , •¬ and Takeshi NISHINO
Department of Microbiology, Kyoto Pharmaceutical University,
Kyoto, Kyoto 607, Japan
(Accepted for publication, September 25, 1991)
Abstract The antibacterial activity of cefpodoxime against Branhamella catarrhalis

was studied. All of the 65 clinical isolates tested were inhibited at and below 1.56
μg/ml, both at 107 and at 105 CFUs. The following was further studied on B.
catarrhalis N-5 which showed average susceptibility to each drug examined. Bac-
tericidal activity was observed at and above the MIC. Scanning and transmission
electron microscopy revealed morphological changes, such as cellular swelling,
bleb formation, inhibition of septum formation, and lysis, of the cells exposed to
cefpodoxime at concentrations around the MIC. Cefpodoxime was poorly hydro-
lyzed by the ƒÀ-lactamase and it showed affinity for two penicillin-binding proteins
that had approximate molecular weights of 83 and 74 kilodaltons, with I50 values
of 3.7 and 2.1 ƒÊg/ml, respectively.
Moraxella subgenus Branharnella catarrhalis (B. catarrhalis), an oropharyngeal

commensal, has been increasingly recognized as a pathogen that causes mainly
acute otitis media, acute maxillary sinusitis, and bronchopulmonary infections

,
particularly in patients with underlying chronic pulmonary disease (4, 6). During
the last decade the occurrence of respiratory tract infections due to B . catarrhalis has
been reported worldwide (6), including Japan (25). The organism has become
one of the most frequently isolated bacteria among respiratory pathogens, along
with Haemophilus influenzae and Streptococcus pneurnoniae (20

, 28).
In 1977, ƒÀ - lactamase production in clinically significant isolates of B . catarrhalis
was first reported (18, 24). Since then the proportion of ƒÀ -lactamase - producing
strains has increased to nearly 90% in some areas (2

, 30). Treatment with penicil-
lins and some cephalosporins has occasionally been ineffective against these strains
(12, 20, 24, 25).
Cefpodoxime - proxetil is an orally active oxime-type cephalosporin, containing
methoxymethyl at the 3 position and aminothiazolyl in the 7ƒÀ -acyl substituent .
It has been reported that cefpodoxime had a broad spectrum against gram-positive
and -negative bacteria, and that it was stable against ƒÀ -lactamases of many types
,
though it was slightly hydrolyzed by cefuroximases (10
, 31).
〓 Present address: Biological Research Laboratories, Sankyo Co ., Ltd., Shinagawa- ku, Tokyo
140, Japan.
1059
13480421, 1991, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1348-0421.1991.tb01628.x, Wiley Online Library on [18/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1060 T. TAKENOUCHI AND T. NISHINO
Cefpodoxime-proxetil is admitted to use for B. catarrhalis infections, though
preceding oral ƒÀ -lactams, such as cephalexin, cefadroxil, cefaclor, cefteram-pivoxil,

and amoxicillin, are not. We examined the antibacterial activity of cefpodoxime,
the active metabolite of cefpodoxime -proxetil, against B. catarrhalis.
MATERIALS AND METHODS
Antibiotics. Cefpodoxime was donated by Sankyo Co., Ltd., Tokyo. Cepha-
lexin and cefaclor were obtained from Shionogi & Co., Ltd., Osaka, Japan, cefa-
droxil from Nippon Bristol Co., Ltd., Tokyo, cefteram from Toyama Chemical Co.,
Ltd., Tokyo, cephaloridine from Nippon Glaxo Co., Ltd., Tokyo, penicillin G
from Meiji Seika Kaisha Ltd., Tokyo, and amoxicillin from Kyowa Hakko Kogyo
Co., Ltd., Tokyo.

Bacterial strains. The 19 clinical isolates of B. catarrhalis employed in this
study were generously provided by Dr. K. Matsumoto, the Department of Internal
Medicine, Institute for Tropical Medicine, Nagasaki University, Nagasaki, Japan.

The other 46 isolates were kindly provided from Dr. K. Shimada, Department of
Infectious Disease, Institute of Medical Science, The University of Tokyo, Tokyo.
Fifty- five of the 65 strains (88%) were shown to be ƒÀ- lactamase producers by nitro-
cefin color test (22).
Susceptibility test. MICs were determined by a twofold serial dilution method

using heart infusion agar (Nissui) supplemented with 5% chocolatized horse blood.
With an inoculator device, one loopful of bacterial suspension (ca. 10 ƒÊl), containing
approximately 107 and 105 CFUs, was inoculated onto the agar plates. The plates
were incubated aerobically at 37 C for 20 hr, and the MICs were taken as the
lowest concentrations of the drugs that inhibited the visible growth of bacteria.
Effect on growth. Overnight culture of B. catarrhalis N-5 in heart infusion broth
(HIB; Nissui) was diluted 1,000-fold with trypto- soy broth (TSB; Nissui) and
incubated at 37 C with shaking. When the growth reached a level of 106 or 107
CFU/ml, cefpodoxime was added to the cultures at various concentrations around
the MI C. Subcultures were further incubated at 37 C with shaking, and portions
were sampled at 0, 2, 4, and 6 hr, respectively. After serial 10-fold dilution with
saline, 0.1 ml of each dilution was plated on nutrient agar (Difco) supplemented
with 5% horse blood, to determine the residual CFUs.

Electron microscopic observation. B. catarrhalis N-5 cells were incubated in TSB
with shaking as described above, and when the bacteria reached the logarithmic
phase of growth (OD550•à 70.5), cefpodoxime was added. Portions were sampled
at 0, 1, 2, and 4 hr after addition of the antibiotic, and samples were double-fixed
with glutaraldehyde and osmium tetroxide according to the method of Kellenberger
et al (16). The fixed samples were further processed for electron microscopy as
described previously (21); they were examined using a scanning electron micro-
scope (JSM-35; JEOL, Tokyo) at 20 kV acceleration and a transmission electron
microscope (JEM- 1200EX; JEOL) at 80 kV.
Assay of ƒÀ -lactamase. An overnight culture in TSB (5 ml) was inoculated into

ACTIVITY OF CEFPODOXIME AGAINST B. CATARRHALIS 1061
1,000 ml of HIB and the cells were grown at 37 C with shaking. The late-loga-
rithmic-phase cells were harvested by centrifugation in a refrigerated centrifuge at
10,000 •~g for 10 min, followed by one washing with ice-cold 10 mm sodium phos-
phate buffer (pH 7.0). The cells were suspended in 25 ml of 50 mm sodium phos-
phate buffer (pH 7.0) and disrupted with an ultrasonicator 18 times for 30 sec on
ice. Unbroken cells were removed by centrifuging at 6,000 •~g for 10 min at 4 C,
and the supernatant was further centrifuged at 100,000 •~g for 45 min. The resulting
supernatant was stored at -80 C and used as a crude extract of ƒÀ - lactamase. ƒÀ-
Lactamase activity was determined by the spectrophotometric method of Okonogi
et al (23). The specific wavelength and the molar differential absorbance co-
efficient used for cefpodoxime were 265 nm and 6,750 m-1•Ecm-1, respectively. The
maximum rate of hydrolysis (Vmax) and Michaelis constant (Km) were estimated
from Lineweaver-Burk plots (17), and substrate specificity was expressed as relative
Vmax, taking the value of penicillin G as 100.
Assay of PBPs. The membrane fraction was prepared by ultrasonic treatment,
followed by centrifuging as mentioned above. The precipitate was then washed
once with 50 mm sodium phosphate buffer and suspended in 50 mm sodium phosphate
buffer containing 10 mm MgCl2 at a final protein concentration of 20 mg/ml. Com-
petitive binding of drugs to penicillin-binding proteins (PBPs) was assayed by the

method of Spratt (27). Briefly, membrane samples were preincubated with several
concentrations of drugs for 10 min at 30 C, and the PBPs still accessible were mea-
sured by the addition of 14 C-labeled penicillin G (0.3 ƒÊCi; potassium 6-phenyl
[1-14C] acetamidopenicillanate, Amersham International, Buckinghamshire, U.K.)

for another 10 min at 30 C. The 0.7% (NOT) sarcosyl-soluble fractions were
separated on a 7.5% polyacrylamide slab gel. After exposure to X-ray film for
10 wk at -80 C, the fluorograph of the PBPs was quantitatively analyzed by a
laser densitometer (ULTROSCAN XL; LKB) and 150 (concentration required to
inhibit [14C]penicillin G binding by 50%) was calculated. Molecular weights of
the PBPs were estimated from a mobility versus logarithm of the molecular weight
curve constructed from known PBPs of Escherichia coli K12 as standard (27).
RESULTS
Drug Susceptibility
The susceptibilities of 65 clinical isolates to cefpodoxime and its related drugs
are presented in Table 1. Cefpodoxime inhibited the growth of all the strains
examined at and below the concentration of 1.56 ƒÊg/ml, both at 107 and at 105
CFUs, and it was more active than the other drugs tested. A reasonably narrow
range of MICs (<16-fold difference) was observed with cefpodoxime. Broader
variations were, on the other hand, observed with the other drugs, especially with
cefaclor and amoxicillin at 107 CFUs.
Effect on Growth
The effect of cefpodoxime on the viability of B. catarrhalis N-5 is shown in

Table 1. Susceptibility of 65 clinical isolates to cefpodoximea)
A B
Fig. 1. Effect of cefpodoxime on the viability of B. catarrhalis N-5. The drug was added to
cultures containing 107 (A) and 106 (B) CFU/ml of bacterial cells at concentrations of
0.20 (○---- 〇), 0.39(▲-- ▲), 0.78 (△- △), 1.56 (■-・- ■), and 3.13μg/ml(□-・・-□).
MIc at 106 cFu/ml: 0.78 μg/ml. control (●- ●).
Fig. 1. B. catarrhalis N-5 is a ƒÀ -lactamase- producing strain that showed average
susceptibility to each drug among the 65 strains tested. Bactericidal activity was
observed at and higher than the MIC (0.78 ƒÊg/ml) when the drug was added to
the cultures containing 106 CFU/ml of bacterial cells. A similar result was ob-
tained during the first 2 hr when the drug was added to 107 CFU/ml of cultures.
Thereafter regrowth occurred, however, at concentrations of 1- and 2- fold the MIC
of cefpodoxime, while reduction in viability was still demonstrated at a concentration
of 4- times the MIC. Residual activity of cefpodoxime in the culture broth de-
creased from the initial concentration of 0.78 ƒÊg/ml to a level undetectable (•¬0.10
μg/ml) by bioassay after a 3-hr incubation.

ACIIVII OF CEFPODOXIME AGAINST B. CATARRHALIS 1063
A B
C D
Fig. 2. Scanning electron micrographs of B. ,atarthaUs N-5 exposed to 0.78 ƒÊg/ml of celpo-
doxime for 0 (A), 1 (114), 2 (C), and 4 hr (D). Cellular swelling and bleb formation were
observed. •~ 10,000. Bars, 1 ƒÊn.
Scanning Electron Microscopic Observation
Figure 2 shows serial observations of the surface structure of bacterial cells
after exposure to 0.78 ƒÊg/ml of cefpodoxime. Untreated 13. catarrhalis N-5 cells
exhibited smooth surfaces (Fig. 2A). Figure 2B shows cells at 1 hr after exposure,
showing cellular swelling and prominent bleb formation. Prolonged exposure to
the drug resulted in an increase in cellular swelling with variations in size, and
apparently decreased bleb formation which would have resulted from the lysis of
bleb -fbrming cells (Fig. 2, C and D). A similar morphological change with lysis
was observed when cells were exposed to 3.13 ƒÊg/ml of cefpodoxime, and there was
little change at 0.20 ƒÊg/ml.

Fig. 3. Ultrathin section of untreated B. catarthalis N-5 cells. •~ 15,000. Bar, 0.5 ƒÊm.
Membrane proteins were analyzed by sodium dodecyl sulfate - polyacrylamide
gel electrophoresis to determine whether the relative amount of specific proteins
was altered by 1-hr exposure to 0.78 ƒÊg/ml of cefpodoxime, which causcd prominent
bleb formation. No significant difference was observed between the protein profile
of the treated sample and that of the nontreated control (data not shown).
Transmission Electron Microscopic Observation
Figure 3 shows an ultrathin section of untreated B. catarrhalis N-5 cells, which
were dividing and had rather undulate outer membranes. Figure 4 shows cells
exposed to 0.20 ƒÊg/ml of cefpodoxime for 4 hr, with swelling in the septum, sug-
gesting the accumulation of peptidoglycan at the site. Figure 5 shows cells exposed
to 0.78 ƒÊg/ml of the drug for 1 hr, indicating abnormal septum formation (Fig. 5A)
and inhibition of cell division exemplified by rod-like cells (Fig. 5B). Figure 6
shows cells exposed to the drug at 3.13 ƒÊg/ml for 1 hr, in which lytic cells (Fig. 6A),
stretched constriction forms, implying inhibition of septum formation at an early

Fig. 4. Ultrathin section of B. catarrhalis N-5 cells exposed to 0.20 ƒÊg/ml of cefpodoxime for
4 hr. Swelling at the septum was observed. •~ 15,000. Bar, 0.5 ƒÊm.
stage (Fig. 6B), and formation of blebs comprising vesicles within them (Fig. 6C)
were found.
Stability and Affinity for ƒÀ-Lactamase
Table 2 shows the relative Vmax and Km of cefpodoxime and its related drugs
for ƒÀ-lactamase produced by B. catarrhalis N-5. All of the compounds examined,
including cefpodoxime, were hydrolyzed to some extent with differences in degree.
Among cephalosporins, cephaloridine and cefaclor showed higher hydrolysis rates
than penicillin G, with relative V. values of 245 and 216, respectively, although
their affinities were comparatively low.
Binding Affinity for PBPs
Figure 7 shows the fluorograph after competition of 7 concentrations of cefpo-
doxime with 14C- labeled penicillin G, and Table 3 gives the approximate molecular
weight and 150 calculated by densitometry for each PBP. Nine major PBPs were
A B
Fig. 5. Ultrathin section of B. catarrhalis N-5 cells exposed to 0.78 ƒÊg/ml of cefpodoxime
1 hr. Abnormal septum formation (A, •~ 18,000) and a rod-like cell (B, 6,000) were
observed. Bars, 0.5 ƒÊm.
detected; their molecular weights ranged from 122 to 41 kilodaltons. (efpodoximc
showed affinities only for PBPs 3 and 4, with respective I50s of 3.7 and 2.1 ƒÊg/ml.
Fifty-percent saturation of the other 7 PBPs required concentrations in excess of
400 ƒÊg/ml, the highest concentration tested.
Affinities of several antibiotics for PBPs were examined under the same assay
conditions. The antibiotics used were cephaloridine, mecillinam, cephalexin, and
cefoxitin, which have been shown to bind different PBPs in E. coli K12 (27). These
β-lactams were also bound to only PBPs 3 and 4. Pretreatment of membrane

samples with clavulanic acid before the addition of 14AC-labeled
penicillin G clarified
all PBPs and revealed another major band, PBP 3' (80 kilodaltons).
DISCUSSION
Many investigators have assessed B. catarrhalis in terms of drug susceptibilities
(1, 2, 5, 7, 18, 24, 25, 28, 29), substrate profiles to ƒÀ- lactamase, and biochemical
properties (8, 9, 13-15, 19, 26, 28, 33), but no work has been done on morpho-
logical alterations of this organism exposed to an antibiotic. In the present study
we examined the effect of cefpodoxime on the morphology of B. catarrhatis by electron
microscopy. The observations showed cellular swelling, bleb formation, inhibition

1067
for
(C.
B
formation
cefpodoxime
B. CATARRHALIS
of
bleb
and
3.13 ƒÊg/ml
C).
C
12,000),
to
and
(B
AGAINST
exposed
(13,
0.5 ƒÊm
cells
constriction
and
N-5
CEFPODOXIME
(A)
catarrhalis
in
stretched
I
B.
Bars,
of
OF
5,000),
section
found.
ACTIVITY
(A,
were.
Uhrathin
Lysis
,000)
hr.
×15
Fig. 6.
I
Fig. 7. Fluorograph showing competition of cefpodoxime with 14C-labeled penicillin C

binding in B. catarrhalis N-5.
Table 2. Hydrolysis of cefpodoxime to ƒÀ -lactamase from
B. catarrhalis N-5
Table 3. Competition of cefpodoxime with He-labeled penicillin G

for binding to PBPs in B. catarrhalis N-5
Abbreviations: CPDX, cefpodoxime: CER, cephaloridine; MPC, mecillinam;

CEX, cephalexin: (TX, cefoxitin.
ACTIVITY OF CEFPODOXIME AGAINST B. CATARRHAM 1069
of septum formation, and lysis. The relationship between morphological changes
and the function of PBP(s) still remains unknown in this species.
As fatal infections are rarely caused by B. catarrhalis, it is sufficient to treat most
patients with oral antibiotics (1). Cefpodoxime belongs to a class of third- genera-
tion cephalosporins based on its chemical structure and antibacterial spectrum
(10, 31). Cefpodoxime inhibited all the test strains up to the concentration of
1.56 ƒÊg/ml, substantially without regard to inoculum size. Amoxicillin and cefaclor
showed profound inoculum effects in MICs (8-fold or more), in accord with previous
observations (8, 24, 30). Added to this is etiological evidence that B. catarrhalis is
frequently isolated in combination with H. influenzae or S. pneumoniae or both (6, 20,
24, 25), against which cefpodoxime is more active than earlier oral ƒÀ-lactams
(10, 31).
The ƒÀ-lactamase of B. catarrhalis has been shown to be different from other
β- lactamases in gram-negative bacteria regarding substrate profiles, inhibition rates,

and isoelectric focusing patterns (14, 28, 33), and is classified as group 2C according
to Bush's classification (3), which hydrolyzes carbenicillin and is inhibited by
clavulanic acid. Some deviations exist among Branhamella ƒÀ - lactamases (9, 19,
28, 32) ; they hydrolyze some cephalosporins as well as penicillins. The substrate
profile to ƒÀ-lactamase from B. catarrhalis N-5 seems to be rather unique as com-
pared to previous reports. Cephaloridine was hydrolyzed at a higher rate than
penicillin G. This was seen only in satellite bands of strain 2001E (26) and one
strain out of 57 isolates in this experiment (data not shown). However, some
authors have pointed out the instability of cefaclor against ƒÀ-lactamase, especially
at an elevated pH (8, 13, 14). Further investigations of strains and characterizations
should be undertaken.
As is rarely the case with cocci, fluorography of B. catarrhalis N-5 showed 9
major radioactive bands, with many minor ones. Georgopapadakou et al (11)
reported that Triton X- 100 extraction from one strain revealed 3 PBPs after 20-day
exposure, and 77- and 39-kilodalton PBPs were insensitive to cephalothin (no
description about 68-kilodalton PBP). These PBPs seemed to be identical to
PBP 3', 5, and 9 in this study, considering that these components mainly appeared
in one ƒÀ-lactamase-negative strain (data not shown). The difference in number
would simply result from a longer exposure period of 10 wk, because [14C]penicillin
G was partially hydrolyzed by ƒÀ -lactamase during the assay. Clarification of
PBPs by pretreatment with clavulanic acid indicates tight binding of ƒÀ-lactamase
to the membrane (8, 9) and high affinity with this compound (9, 18, 32, 33), as
other investigators have described. It seems necessary for detecting PBPs of [3-
lactamase -producing B. catarrhalis to expose the gel for longer period than usual,
or pretreat with clavulanic acid.
In competitive binding assay, cefpodoxime exhibited affinities only to PBPs
3 and 4, with approximate molecular weights of 83 and 74 kilodaltons, respectively.
On the basis of similar findings obtained with several ƒÀ- lactams, it can be concluded
that these two PBPs are the target proteins. It is not clear from the results of this
experiment, but PBP 3 of B. catarrhalis N-5 would correspond to PBPs 2 and 3 of

E. coli K12, because mecillinam and cephalexin showed affinity solely for this
protein.
We thank Dr. S. Ohya, Biological Research Laboratories, Sankyo Co., Ltd., for constructive
suggestions in preparation of the manuscript.
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Microbiology and Immunology - December 1991 Ouchi - Antibacterial Activity of Cefpodoxime Against Branhamella

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Microbiology and Immunology - December 1991 Ouchi - Antibacterial Activity of Cefpodoxime Against Branhamella

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13480421, 1991, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1348-0421.1991.tb01628.x, Wiley Online Library on [18/01/2023].

Antibacterial Activity of Cefpodoxime against

Takashi TAKENOUCHI, * , •¬ and Takeshi NISHINO

Department of Microbiology, Kyoto Pharmaceutical University,

Kyoto, Kyoto 607, Japan

(Accepted for publication, September 25, 1991)

Abstract The antibacterial activity of cefpodoxime against Branhamella catarrhalis

electron microscopy revealed morphological changes, such as cellular swelling,

cefpodoxime at concentrations around the MIC. Cefpodoxime was poorly hydro-

of 3.7 and 2.1 ƒÊg/ml, respectively.

Moraxella subgenus Branharnella catarrhalis (B. catarrhalis), an oropharyngeal

acute otitis media, acute maxillary sinusitis, and bronchopulmonary infections

with Haemophilus influenzae and Streptococcus pneurnoniae (20

strains has increased to nearly 90% in some areas (2

(12, 20, 24, 25).

Cefpodoxime - proxetil is an orally active oxime-type cephalosporin, containing

methoxymethyl at the 3 position and aminothiazolyl in the 7ƒÀ -acyl substituent .

Cefpodoxime-proxetil is admitted to use for B. catarrhalis infections, though

preceding oral ƒÀ -lactams, such as cephalexin, cefadroxil, cefaclor, cefteram-pivoxil,

the active metabolite of cefpodoxime -proxetil, against B. catarrhalis.

MATERIALS AND METHODS

Antibiotics. Cefpodoxime was donated by Sankyo Co., Ltd., Tokyo. Cepha-

Co., Ltd., Tokyo.

study were generously provided by Dr. K. Matsumoto, the Department of Internal

Medicine, Institute for Tropical Medicine, Nagasaki University, Nagasaki, Japan.

Infectious Disease, Institute of Medical Science, The University of Tokyo, Tokyo.

cefin color test (22).

Susceptibility test. MICs were determined by a twofold serial dilution method

Effect on growth. Overnight culture of B. catarrhalis N-5 in heart infusion broth

CFU/ml, cefpodoxime was added to the cultures at various concentrations around

the MI C. Subcultures were further incubated at 37 C with shaking, and portions

with 5% horse blood, to determine the residual CFUs.

with glutaraldehyde and osmium tetroxide according to the method of Kellenberger

scope (JSM-35; JEOL, Tokyo) at 20 kV acceleration and a transmission electron

microscope (JEM- 1200EX; JEOL) at 80 kV.

Assay of ƒÀ -lactamase. An overnight culture in TSB (5 ml) was inoculated into

rithmic-phase cells were harvested by centrifugation in a refrigerated centrifuge at

Lactamase activity was determined by the spectrophotometric method of Okonogi

Vmax, taking the value of penicillin G as 100.

Assay of PBPs. The membrane fraction was prepared by ultrasonic treatment,

followed by centrifuging as mentioned above. The precipitate was then washed

once with 50 mm sodium phosphate buffer and suspended in 50 mm sodium phosphate

buffer containing 10 mm MgCl2 at a final protein concentration of 20 mg/ml. Com-

petitive binding of drugs to penicillin-binding proteins (PBPs) was assayed by the

sured by the addition of 14 C-labeled penicillin G (0.3 ƒÊCi; potassium 6-phenyl

[1-14C] acetamidopenicillanate, Amersham International, Buckinghamshire, U.K.)

10 wk at -80 C, the fluorograph of the PBPs was quantitatively analyzed by a

laser densitometer (ULTROSCAN XL; LKB) and 150 (concentration required to

inhibit [14C]penicillin G binding by 50%) was calculated. Molecular weights of

The susceptibilities of 65 clinical isolates to cefpodoxime and its related drugs

range of MICs (<16-fold difference) was observed with cefpodoxime. Broader

cefaclor and amoxicillin at 107 CFUs.

The effect of cefpodoxime on the viability of B. catarrhalis N-5 is shown in

Table 1. Susceptibility of 65 clinical isolates to cefpodoximea)

MIc at 106 cFu/ml: 0.78 μg/ml. control (●- ●).

Fig. 1. B. catarrhalis N-5 is a ƒÀ -lactamase- producing strain that showed average

Thereafter regrowth occurred, however, at concentrations of 1- and 2- fold the MIC

of cefpodoxime, while reduction in viability was still demonstrated at a concentration

μg/ml) by bioassay after a 3-hr incubation.

observed. •~ 10,000. Bars, 1 ƒÊn.

Scanning Electron Microscopic Observation

Figure 2 shows serial observations of the surface structure of bacterial cells

showing cellular swelling and prominent bleb formation. Prolonged exposure to