Vol. 34, No. 5
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1990, p. 813-818
0066-4804/90/050813-06$02.00/0 Copyright X 1990, American Society for Microbiology
In Vitro Activity of Bay v 3522, a New Cephalosporin, Compared with Activities of Other Agents R. WISE,* J. M. ANDREWS, J. P. ASHBY, AND D. THORNBER Department of Medical Microbiology, Dudley Road Hospital, Birmingham B18 7QH, United Kingdom Received 14 November 1989/Accepted 8 February 1990
The in vitro activity of Bay v 3522, a new aminobenzothiazol cephem, was compared with those of other oral ,-lactams. Bay v 3522 displayed high activity against Staphylococcus spp. (MICs for 90% of strains tested [MIC90s], 0.5 ,g/ml), Streptococcus pneumoniae (MICgo, 0.06 ,ug/ml), and Haemophilus influenzae and BranhameUla catarrhalis (MIC90s, 2 pg/ml). There was limited activity against members of the family Enterobacteriaceae, with the NIIC9s being between 4 and >128 pg/ml. The stability of Bay v 3522 to hydrolysis by the SHV-1 and TEM-1 enzymes was intermediate to those of cephalexin (least hydrolyzed) and cefaclor, but it was markedly more stable than amoxiciflin. There was high affinity to the chromosomally mediated P99 enzyme. The protein binding of Bay v 3522 was 45%. The primary target of Bay v 3522 was penicillin-binding protein 3.
Bay v 3522 is a new oral cephem agent which has the formula 7-[D-2-amino-2-(2-aminobenzothiazol-6-yl-acetoamide]-3-[(Z)-l-propen-l-ly]-3-cephem-4-carboxylic acid monohydrate. Preliminary information (Bayer AG, Wuppertal, Federal Republic of Germany) suggests that this compound has high activity against staphylococci and Haemophilus influenzae. In this study, the activity of Bay v 3522 was compared with those of other oral cephalosporins, amoxicillin-clavulanate, and other relevant agents.
transferred to the surface of the antibiotic-containing agar with a multipoint inoculating device (Denley-Tech, Billingshurst, England). The final inocula on the plates were therefore 104 and 106 CFU. The medium used for the agar dilution procedure was Iso-Sensitest agar (pH 7.2; Oxoid), which was supplemented as follows: 5% horse blood plus 1% supplement C was added to support growth of streptococci, Haemophilus influenzae, and Neisseria spp.; for anaerobes, Wilkins-Chalgren agar was used. All plates were incubated in air at 37°C for 24 h, except for the following. The anaerobes were grown in an anaerobic cabinet in an atmosphere of 10% hydrogen-10% carbon dioxide-80% nitrogen; Haemophilus influenzae and Neisseria spp. were incubated in air enriched with 6% carbon dioxide. In addition, Staphylococcus aureus was also incubated in air at 30°C, with 5% sodium chloride added to the medium. The MIC of the antibiotic was defined as that concentration (in micrograms per milliliter of agar) at which no more than two colonies were detected. In the case of the higher inoculum, a slight haze of growth was ignored. The effect of human serum on the MIC and MBC of Bay v 3522 was studied with eight strains (two each of Klebsiella spp., Escherichia coli, Branhamella catarrhalis, and Staphylococcus aureus) by a method based on that of Pearson et al. (8); the bactericidal endpoint was 99.9% lethality. An overnight broth culture of these organisms was inoculated into 1 ml of Iso-Sensitest broth with 20 and 70o human serum and decreasing concentrations of the antimicrobial
MATERIALS AND METHODS A total of 561 strains were tested, of which 554 were recent clinical isolates from different patients. The remaining strains were well characterized ,-lactamase producers. The following antibiotics were obtained from the indicated sources: Bay v 3522, Bayer AG; cefixime, Lederle Laboratories (Fareham, England); cefuroxime and cephalexin, Glaxo Research Laboratories (Greenford, England); amoxicillin, penicillin, and clavulanic acid, Beecham Research Laboratories (Brentford, England); and erythromycin, Abbott Laboratories (Maidenhead, England). Susceptibility testing. The susceptibilities of the strains were studied by a routine agar plate dilution method. Amoxicillin and clavulanate were combined in a 2:1 ratio, and the results were expressed in terms of the amoxicillin MIC. The inocula were prepared as follows. For all strains except streptococci (including Streptococcus pneumoniae), Neisseria spp., Haemophilus influenzae, and anaerobes, the organisms were grown overnight in nutrient broth to yield a viable count of about 109 CFU/ml. Streptococci, Haemophilus influenzae, and Neisseria spp. were grown in brain heart infusion broth (Oxoid Ltd., Basingstoke, England) plus 1% supplement C (Difco, East Molesley, England). Bacteroides spp. were grown in Wilkins-Chalgren broth (Oxoid Ltd.) plus 0.25% sodium succinate. Clostridia were grown in Wilkins-Chalgren broth supplemented with 1% Tween 80 (which was previously shown to enhance growth). The viable counts were comparable in each broth culture. One microliter of a 1:1,000 dilution of an overnight culture or, for a few selected strains upon which an increased inoculum was to be studied, an undiluted inoculum was *
agent.
The protein binding of Bay v 3522 was estimated in triplicate in human serum by an ultrafiltration technique with a Centriflo cone (Amicon Corp., Lexington, Mass.) with an exclusion limit of 50,000 daltons. The concentrations of Bay v 3522 used were 1 and 25 ,ug/ml. The ultrafiltrate (after pH adjustment to precentrifugation values with CO2 gas) was assayed by a microbiological method against standards prepared in phosphate buffer at pH 6.5 (the pH of the ultrafiltrate). The indicator organism was Morganella morganii 313UC3 (from Schering Corp., Bloomfield, N.J.), and the medium was antibiotic medium no. 2 (Oxoid Ltd.). ,i-Lactamase stability. To determine ,-lactamase stability, six strains were studied: Escherichia coli (SHV-1 producer),
Corresponding author. 813
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Activity of Bay v 3522 compared with those of other agents Organism (no.)
Antibiotic
50%
90%yo
Range
Escherichia coli (50)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
4 0.12 2 4 2
32 0.5 4 16 8
1->128 0.015-4 0.12-64 2->128 0.5-16
Klebsiella spp. (50)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
2 0.03 1 4 1
>128 0.12 16 16 8
0.25->128 0.008-0.5 0.12->128 2->128 0.25-16
Proteus mirabilis (50)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
1 0.008 0.5 8 0.5
4 0.008 2 8 2
Proteus vulgaris (20)
Bay v 3522 Cefixime Cefuroxime
Cephalexin Amoxicillin-clavulanate
>128 0.008 16 128 2
>128 0.015 32 >128 4
32->128 0.008-0.06 1-64 32->128 0.5-32
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
128 0.12 16 128 32
>128 8 128 >128 64
1->128 0.004->128
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
128 0.015
>128 0.12 >128 >128 64
16->128 0.008-0.5 4->128 0.12->128 4-128
Morganella morganii (25)
Providencia stuartii (19)
32 64 32
>128 >128 32
32->128 0.12->128 16->128 0.12->128 4-128
64 8 32 >128 8
128 32 64 >128 32
8-128 1-64 4->128 16->128 2-128
>128
Acinetobacter spp. (20)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
Staphylococcus aureus (31) (including 3 methicillin-resistant strains)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate Erythromycin
0.25 8 0.5
Staphylococcus epidermidis (29)
Bay v 3522 Cefixime Cefuroxime Cephalexin AmoxicilHin-clavulanate Erythromycin
Staphylococcus saprophyticus (30)
Bay v 3522 Cefixime Cefuroxime
marcescens
[5];
0.12->128 1->128 1-64
>128 128 >128 >128 128
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
Serratia spp. (10) (Serratia Serratia liquefaciens [5])
1-16 0.008-0.06 0.25-16 4-128 0.5-16
1
2 0.25 0.12
0.5 16 2 8 0.5 >128
0.12-16 2-128 0.25->128 0.25-64 0.064 0.06->128
0.12 4 0.25 0.5 0.12 0.06
0.5 16 2 8 0.5 >128
0.06-0.5 4->128 0.12-2 0.5-8 0.03-0.5 0.06->128
0.25 32 2
O.5 64 2
0.06-16 4-128 0.25->128
Continued on following page
IN VITRO ACTIVITY OF Bay v 3522
VOL. 34, 1990
815
TABLE 1-Continued Organism (no.)
Antibiotic
5O0o
Cephalexin Amoxicillin-clavulanate
Erythromycin Enterococcus faecalis (20)
Bay v 3522 Cefixime Cefuroxime
Cephalexin Amoxicillin-clavulanate Erythromycin
2 0.25 0.12
2 32 32 128 0.5 1
90%
8 0.5 0.25 4 >128 >128 128 0.5 >128
Range 1-128 0.03-16 0.06-128 2-4 4->128 8->128 64-128 0.25-0.5 0.5->128
Streptococcus pneumoniae (28)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate Erythromycin Penicillin
Bacteroides fragilis (14)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
Branhamella catarrhalis (29)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
1 0.06 1 4 0.06
2 0.5 2 4 0.25
0.12-2 0.03-1 0.25-2 2-8 0.015-0.5
Neisseria gonorrhoea (19) (including 3 ,3-lactamase-producing strains)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
0.5 0.004 0.03 2 0.12
4 0.008 0.12 4 0.5
0.015-4 0.004-0.015 '0.002-0.12 0.5-16 0.008-1
Neisseria meningitidis (10)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate
0.06 0.004 0.03 1 0.06
0.12 0.004 0.03 1 0.12
0.06-2 0.004-0.6 0.015-1 0.5-2 0.03-0.12
Haemophilus influenzae (33) (including 12 P-lactamase-producing strains)
Bay v 3522 Cefixime Cefuroxime Cephalexin Amoxicillin-clavulanate Amoxicillin
a
0.03 0.12 0.03 2 0.015 0.12 0.015 8 16 8 32 16
1 0.03 1 16 1 0.5
0.06 0.25 0.06 2 0.03 16 0.03
128 >128 128 >128 32
2 0.12 2 64 2 8
0.008-0.5 0.03-2 0.004-0.25 0.5-16 0.008-0.25 0.06-16 0.004-0.5 2->128 4->128 1->128 16->128 0.06->128
0.5-16 0.008-2 0.25-32 4->128 0.25-16 0.25-16
50%7o and 90o, MICs for 50 and 90%o of strains tested, respectively.
Staphylococcus aureus NCTC 1156, Klebsiella aerogenes (K-1 producer provided by Glaxo Pharmaceuticals, Ltd., Greenford, United Kingdom), Enterobacter cloacae (P99 producer provided by N. Curtis), and two clinical isolates (Branhamella catarrhalis [BRO-1] and Haemophilus influenzae [TEM-1]). Cell-free sonic extracts were prepared from mid-exponential-phase cultures in tryptone soy broth as described previously (1). Haemophilus influenzae was cultured on basic medium supplemented with 25 ,ug of NAD per ml and 5% horse blood. The subsequent colonies were scraped from the surfaces of approximately 200 plates, suspended in 15 ml of ice-cold phosphate buffer (pH 7), and then subjected to the process of sonication and centrifugation described previ-
ously (1). Staphylococcal penicillinase was prepared as described previously (3). The extracts were assayed for protein by the method of Lowry et al. (5), and isoelectric focusing was carried out on polyacrylamide gel plates (LKB Instruments, Inc., Rockville, Md.) with an electrophoretic unit (Multiphor II; LKB Instruments) with a constant 2.5-kV constant power supply. Hydrolysis studies were carried out on amoxicillin, cephaloridine, cefaclor, cephalexin, and Bay v 3522 in a temperature-controlled UV spectrophotometer (Lambda 2; PerkinElmer, Beaconsfield, United Kingdom) at 37°C (7). All antibiotic solutions were prepared immediately before use in 50 mM sodium phosphate buffer, and the reaction was followed at that wavelength which gave the maximum
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Susceptibilities of miscellaneous organisms to Bay v 3522 and cephalexin MIC (Oag/ml) Cephalexin Bay v 3522
Streptococcus millerii Enterobacter faecium
0.122, 0.253 82, >1283
Streptococcus sanguis
0.123, 0.6
Streptococcus mitis
0.015, 0.06, 0.252, 8 0.0154, 0.03 0.063, 0.122
0.5, 1, 82, >128
0.015, 0.03, 0.122, 2 0.25, 1, 2 0.015, 0.032, 12, 4
1, 23, 32
Streptococcus pyogenes Lancefield group B streptococci Clostridium perfringens
Clostridium difficile Peptococcus spp. Peptostreptococcus
Proteus rettgeri Salmonella spp. Citrobacter spp. Enterobacter spp.
spp.
22, 42, 8 1282, >1283
45
0-5s
42, 64
0.12, 0.25, 0.5 0.5, 23, 64 24, 8 1, 16, 21283 22, 16, .1282
2, 4, 82, 32 44, 8 4, 16, 64, .1282 4, 8, 16, .1282
a The subscript indicates the number of strains for which the MIC indicated.
was as
change in absorbance before and after hydrolysis. The spectral parameters used in this study were as follows: for cephaloridine and cephalexin, 260 nm; for cefaclor, 265 nm; for amoxicillin, 250 nm; and for Bay v 3522, 274 nm. The reported kinetic constants were derived by the halftime analysis of single-reaction progress curves. For slowly hydrolyzed compounds, affinities were determined by the competing substrate approach and the best estimate of Vmax calculated from initial-rate experiments (4, 6). The relative Vmax rates for Bay v 3522, cephalexin, cefixime, cefaclor, and amoxicillin were compared with that of cephaloridine, which was taken to be 100%. The value calculated for Vm I/Km can be taken as a measure of the efficiency of hydrolysis, as defined by Pollock (9). The specific enzyme activity of the crude ,B-lactamase extract was determined spectrophotometrically by using 0.1 mM cephaloridine as the assay substrate. The enzymatic unit is the amount of enzyme which hydrolyzes 1 p.mol of cephaloridine per min at 37°C and pH 7. The specific activity has been defined as the number of enzyme units per milligram of protein of a given extract. The penicillin-binding protein (PBP) affinity and morphological response to Bay v 3522 were studied in Escherichia coli KL-12. Bacterial envelopes were prepared, and the TABLE 3. Activities of Bay v 3522, cephalexin, and amoxicillin against strains possessing known P-lactamases MIC (,ug/ml)
Enterobacter cloacae 1051E Escherichia coli I153 Escherichia coli 1723E Escherichia coli I386 Klebsiella pneumoniae 1976E Escherichia coli 2138E Escherichia coli 1894E
P99 TEM-1 TEM-2 TEM-5 SHV-1 OXA-1 OXA-3
Bay
SHV-1 TEM-1 K-1 P99
Staphylococcus aureus a The
25
0.033, 0.062
Lactamase
,3-Lactamase
BRO-1
0.52, 1, 324 0.5s, 1, 2, 4
Organism
TABLE 4. Relative efficiency of hydrolysisa
v
Cepha- Amoxi-
3522
lexin
cillin
>128 16 128 64 4 4 4
>128 4 8 4 4 8 4
>128 >128 >128 >128 32 >128 >128
Vmax/Km (%) Bay v 3522 Cephalexin Cefaclor Amoxicillin
8.6 15.6 21 6 26