DIAGN MICROBIOLINFECTDIS 1990;13:349-352
In vitro Activity of BAY v 3522, a N e w Oral Cephalosporin Tested Against Haemophilus influenzae and
Branhamella catarrhalis Gary V. Doern and Tracey A. Tubert
The in vitro activity of BAY v 3522, a new orally absorbed cephalosporin, was assessed against 150 clinical isolates each of Haemophilus influenzae and Branhamella catarrhalis. The MIC9os for beta-lactamase-positive and -negative strains of H. influenzae were 8 and 2 I.~g/ml, respectively. For betalactamase-positive and -negative strains of B. catarrhalis, the BAY v 3522 MICgos were 4 and 0.25 i~g/ml, respectively. In
general, BAY v 3522 was less active against H. influenzae than amoxicillin/clavulanic acid and cefixime, equivalent in activity to cefuroxime, and more active than cefaclor. BAY v 3522 had activity most similar to cefuroxime and cefaclor for B. catarrhalis but was less active than amoxicillin/clavulanaic acid and cefixime.
Bay v 3522 is a new orally absorbed cephalosporin with a (z)-propenyl side chain at the 3 position and a D2(aminobenzothiazol-6-yl) glycyl substituent at the 7 position. Its chemical name is 7-[D-2-amino-2-(2-aminobenzothiazol-6-yl )-acetan~d o ]-3-[ ( z )- l-propen- l-yl]3-cephem-4 carboxylic acid, monohydrate. This comp o u n d originally developed in the research laboratories of Bayer AG in Wuppertal, West Germany, has an estimated serum half-life of 1.7 hr, with peak plasma concentrations of 4-5 ~g/ml, following a 250mg oral dose. Because of the potential use of BAY v 3522 in the management of infections such as acute otitis media, acute maxillary sinusitis, and acute purulent exacerbation of chronic bronchitis, an investigation was performed to assess the in vitro activity of the agent against two of the more common bacterial causes of these diseases, Haemophilus influenzae and Branhamella catarrhalis. The activity of BAY v 3522 was
compared to the activity of six other orally administered antimicrobial agents: penicillin, ampicillin, amoxicillindclavulanic acid, cefaclor, cefuroxime, and cefixime.
From the Department of Clinical Microbiologyand Division of Infectious Disease, University of Massachusetts Medical Center, Worcester, Massachusetts. Address reprint requests to: Gary V. Doern, Ph.D., Department of Clinical Microbiology,University of Massachusetts Medical Center, 55 Lake Avenue, North, Worcester, MA 01655. Received March 13, 1990; revised and accepted March 29, 1990. © 1990Elsevier Science Publishing Co., Inc. 655 Avenues of the Americas, New York, NY 10010 0732-8893/90/$3.50
Strains Tested One hundred fifty clinical isolates of H. influenzae (Hi) were examined in this study. Among these, 56 (37.3%) produced beta-lactamase when assessed by using a nitrocefin-chromogenic cephalosporin assay (Montgomery et al., 1979); 94 (62.7%) did not. Of the 56 beta-lactamase-producing strains of Hi, 23 (41.1%) produced type B capsular antigen; 29 (30.1%) of the 94 beta-lactamase-negative strains of Hi were capsular serotype B. A disproportionately high percentage of beta-lactamase-negative strains of Hi with high minimal inhibitory concentrations (MICs) to ampicillin were intentionally included in this study. Specifically, eight beta-lactamase-negative strains with ampicillin MICs of ~ 4.0 ~g/ml were tested; all lacked type B capsular antigen. In addition, 150 recent clinical isolates of Branhamella catarrhalis (Bc) were tested. Among these, 131 (87.3%) produced beta-lactamase.
Antimicrobial Agents BAY v 3522 was provided by Miles Pharmaceuticals (West Haven, CT). Other agents tested included
G.V. Doern and T.A. Tubert
T A B L E 1.
I n v i t r o A c t i v i t y of BAY v 3522 for Haemophilus influenzae Cumulative Percentage of Strains with MIC (~g/ml) of
BetaNo. of Antimicrobial Lactamase Strains Agent 40.008 +
Ampicillin Amox/Clav~ BAY v 3522 Cefaclor Cefuroxime Cefixime Ampicillin Amox/Clav~ BAY v 3522 Cefaclor Cefuroxime Cefixime Ampicillin Amox/Clav~ BAY v 3522 Cefaclor Cefuroxime Cefixime
0.015 0.03 0.06 0.12 0.25
2 14 2 7
17 30 5 55
84 50 14 91
2 7 16 98 100 70 84 93 45 56 75 96 98 100
59 8 41 1 7 99
79 52 72 2 38 100
90 73 85 28 74
100 87 90 53 87
13 13 13 13
Geometric Mean />64 MIC
96 100 93 100
20.3 1.0 1.8 4.9 0.7 0.09 0.4 0.9 0.5 3.2 1.0 0.03
94 100 95 99 100 78 84 93 100 92 95 100
25 25 25 13 25 75 100
75 100 50 100 38 25 38
8.0 8.0 14.0 32.0 16.0 0.4
75 100 100 63 100
"The concentration listed refers to the concentration of amoxicillin, which was, in all cases, twice the concentration of clavulanic acid. bThese 86 strains of H. influenzae had ampicillin MICs of 2.0 ~g/ml or less. q~hese 8 strains of H. influenzae had ampicillin MICs of 4.0 ~g/ml or greater.
p e n i c i l l i n , a m p i c i l l i n , a m o x i c i l l i n / c l a v u l a n i c a c i d , cefaclor, c e f u r o x i m e , a n d c e f i x i m e . A l l of t h e c o m pounds were obtained from their respective manufacturers as laboratory grade powders.
Broth Microdilution Tests
Broth microdilution trays containing serial twofold c o n c e n t r a t i o n i n c r e m e n t s of a n t i m i c r o b i a l a g e n t s , r a n g i n g f r o m 0.004 to 128 ~ g / m l , w e r e p r e p a r e d b y u s i n g 9 6 - w e l l p l a s t i c t r a y s w i t h a Q u i c k S p e n s e II m u l t i c h a n n e l r e a g e n t d i s p e n s e r ( S a n d y S p r i n g s Instrument Company, Germantown, MD). Haemophilus Test Medium broth, prepared by the invest i g a t o r s , a s d e s c r i b e d p r e v i o u s l y ( J o r g e n s e n et al., 1989), w a s u s e d for t e s t s w i t h H i . C a t i o n - a d j u s t e d M u e l l e r - H i n t o n b r o t h (Difco L a b o r a t o r i e s , D e t r o i t , MI) w a s u s e d for t e s t s w i t h Bc ( N C C L S , 1988). Stock c u l t u r e s w e r e t h a w e d a n d s u b c u l ~ r e d twice to c h o c o l a t e a g a r (Hi) o r s h e e p b l o o d a g a r p l a t e s (Bc). S u s p e n s i o n s of t e s t o r g a n i s m s w e r e p r e p a r e d i n M u e l l e r - H i n t o n b r o t h to a t u r b i d i t y e q u i v a l e n t to t h a t of a 0.5 M c F a r l a n d s t a n d a r d w i t h t h e a i d of a nephelometer (A-Just Meter, Abbott Diagnostics, N o r t h C h i c a g o , IL). A p p r o p r i a t e d i l u t i o n s of i n i t i a l s u s p e n s i o n s w e r e m a d e i n M u e l l e r - H i n t o n b r o t h to a c h i e v e a f i n a l o r g a n i s m c o n c e n t r a t i o n of - 5 x 105 C F U / m l . T r a y s w e r e s e a l e d w i t h M y l a r f i l m a n d in-
8.0Geometric Mean BAY v 3522 MIC (lag/ml)
t (26) (17) Uo) (24)
0.250.12- "(b 0.060;6 0~2 o.ls 0'8 1'0 2'0 4'o 8'0
AMPICILLIN MIC (gg/ml)
FIGURE 1. Relationship between MICs for ampicillin and BAY v 3522 for 94 beta-lactamase-negative strains of H. influenzae. The y-axis defined the geometric m e a n BAY v 3522 MIC for strains of Hi with a given ampicillin MIC; the brackets indicate the range of BAY v 3522 MICs obtained with strains with the indicated ampicillin MIC; the n u m b e r s in parentheses indicate the n u m b e r of strains examined with a particular ampiciUin MIC.
cubated for 20-24 hr at 35°C in ambient air prior to visual determinations of MICs. The MIC was defined as the lowest concentration of antimicrobial agent tested that yielded no visual evidence of growth of the test organism. All MICs were determined twice. The assigned MIC for a given organism-antimicrobial agent combination was defined as the mean of the duplicate MIC determinations. When the two MICs varied by one twofold concentration increment, the higher MIC was taken as the assigned value. Finally, two quality control (QC) strains of Hi and Bc with known MICs to all seven antimicrobials tested were examined twice daily on each day of testing, once at the beginning and once at the end of each test day's run. In no case did a QC strain yield a MIC greater than two concentration increments above or below expected MIC for that given antimicrobial agent. Among the total of 1050 paired MIC determinations with Hi, 925 (88.1%) of the two MICs were identical; in 116 instances (11.0%), the paired MICs varied by one twofold concentration increment; and in the remaining nine cases (0.9%), the duplicate MICs differed by two twofold concentration increments. With Bc, these values were 967 (92.1%), 78 (7.4%), and 5 (0.5%), respectively. The results of MIC determinations with BAY v 3522 and the six other antimicrobial agents tested in this study against Hi are listed in Table 1. With betalactamase-producing strains of Hi, BAY v 3522 was more active than arnpicillin and cefaclor. It was slightly less active, however, than cefuroxime and amoxicillin/clavulanic acid for these strains and markedly less active than cefixime. An MIC of ~ 8.0 ~g/ml TABLE 2.
defines the susceptible interpretive category for most cephalosporins (NCCLS, 1988). Applying the same criterion to BAY v 3522, 54 of the 56 (96.4%) betalactamase-positive strains of Hi tested would have been defined as susceptible. With beta-lactamase-negative strains of Hi, BAY v 3522 activity was exceeded only by cefixime among the cephalosporins. Bay v 3522 and ampicillin appeared to have very similar activity for beta-lactamase-negative strains of Hi. This relationship was further elucidated by comparing BAY v 3522 and ampicillin MICs for individual strains. As demonstrated in Figure 1, there was a striking concordance between the MICs for these two agents with betalactamase-negative strains of Hi. Among the 94 betalactamase-negative strains of Hi examined in this study, 86 had ampicillin MICs of ~ 2.0 ~g/ml and were therefore considered susceptible to ampicillin. All of these strains were found to be susceptible to BAY v 3522, based on the interpretive criterion of 8.0 ~g/ml. The remaining eight beta-lactamase-negative strains had ampicillin MICs of ~ 4.0 ~g/ml and were therefore defined as being resistant. Six of these eight strains had BAY v 3522 MICs of > 8.0 ~g/ml, indicating lack of susceptibility. Similar observation were made with cefaclor and cefuroxime with these eight strains. All seven of the antimicrobial agents examined in this study appeared to possess greater activity for beta-lactamase-negative strains of Bc than for beta-lactamase-positive isolates (Table 2). With strains in the latter group, BAY v 3522 was slightly less active than cefaclor or cefuroxime and markedly less active than amoxicillinYclavulanate or ce-
In vitro Activity of BAY v 3522 for Branhamella catarrhalis Cumulative Percentage. of Strains with MIC (~g/ml) of
No. of Strains
Pencillin Ampicillin Amox/Clav~ BAY v 3522 Cefaclor Cefuroxime Cefixime Penicillin Ampicillin Amox/Clav~ BAY v 3522 Cefador Cefuroxime Cefixime
2 25 92 4 2 1 73
5 43 99 6 21 35 99
16 57 100 11 82 60 100
1 8 65 2
64 95 95
98 99 99
99 100 100
95 100 90
100 48 11 16
100 100 53
Geometric Mean MIC 5.2 1.0 0.09 2.5 1.0 1.0 0.1 0.02 0.007 0.02 0.2 0.2 0.3 0.02
aThe concentration listed refers to the concentration of amoxicillin, which was, in all cases, twice the concentration of clavulanic acid.
f~ime. However, the vast majority (98.5%) of betalactamase-positive strains of Bc had BAY v 3522 MICs of ~ 8.0 ~g/ml and were therefore considered susceptible. Interestingly, with beta-lactamase-negative strains of Bc, BAY v 3522 appeared to be slightly more active than cefaclor and cefuroxime but less active than cefixime or amoxicillin/clavulanic acid. Resistance was not observed among the antimicrobial agents tested in this study against beta-lactamase-negative strains of Bc.
G.V. Doern and T.A. Tubert
In conclusion, the spectrum of in vitro activity of BAY v 3522 for Hi and Bc was found to be most similar to that of cefaclor and cefuroxime. Resistance to BAY v 3522 among clinical isolations of Hi and Bc was uncommon. This study was supported by Miles Pharmaceutical Company (West Haven, CT). We thank Helen Saillant for secretarial assistance.
REFERENCES Jorgensen JH, Redding JS, Maher LA, Howell AW (1989) Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae. J Clin Microbiol 25: 2105. Montgomery K, Raymondo L, Drew WL (1979) Chromogenic cephalosporin spot test to detect beta-lacta-
mase in clinically significant bacteria. J Clin Microbiol 9:205-207. National Committee for Clinical Laboratory Standards (NCCLS) (1988) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard, M7-T2. Villanova, PA: NCCLS.