- Email: [email protected]
Temocillin: In vitro activity against 734 selected clinical isolates, including β-lactamase-producing strains
DIAGNMICROBIOLINFECTDIS 1984;2:55-63
55
NOTES
Temocillin: In Vitro Activity Against 734 Selected Clinical Isolates, Including -Lactamase-Producing Strains Peter C. Fuchs, Arthur L. Barry, Ronald N. Jones, and Clyde Thornsberry
The in vitro activity of temocillin against 734 clinical isolates was tested by broth microdilution. Good activity was demonstrated against Bnterobacteriaceae and both ff.loctamase-positive and -negative strains of Haemophilus int]uenzae and Neisser/a gonorrhoeoe. There was little to no activity against gram-positive cocci and nonfermenting gram-negative bacilli. Bactericidal activity and effect of inocu/um size on temociliin activity were comparable to that of ticarcillin. Temocillin was stable to commonly encountered [Mactamases and significantly inhibited Richmond-Sykes type 1 enzymes of Enterobacter cloacae.
Temocillin (BRL 17421} is a new semisynthetic penicillin structurally similar to ticarcillin, but differing from ticarcillinby the introduction of a methoxy group at the 6-a position of the ~-lactam ring. This modification has rendered temocillin resistant to ~-lactamases, but less active against Pseudomonas species (Julesand Neu, 1982; Slocombe et al.,1981}. Good in vitro activity against a wide spectrum of other bacteria, including most strains of Enterobacteriaceae, Hoemophilus influenzae,and Neisserio gonorrhoeae, has been reported previously {Bolivar et al.,1982; Greenwood et al., 1982; Lagast and Klastersky, 1982; Plot and Van Dyck, 1982; Van Landuyt et al., 1982; Verbist, 1982; Yogev eta]., 1983}. The present study compares the in vitro activity of temocillin with that of related semisynthetic penicillins {ticarcillinand carbenicillin}, newer cephalosporins {cefotaxime and ceftizoxime), and gentamicin against a broad range of recent clinical isolates. A total of 734 clinical isolates was studied. The 178 gram-positive coccal isolates i n c l u d e d 60 strains of Staphylococcus aureus, 10 of w h i c h were methicillin resistant a n d 27 of w h i c h p r o d u c e d ~-lactamase, but were methicillin susceptible; 24 strains of S t a p h y l o c o c c u s epidermidis, 14 of w h i c h were p e n i c i l l i n resistant; 25 strains of Streptococcus faecolis; 26 strains of Streptococcus pneumonioe; 21 strains of Strep-
From the Department of Pathology, St. Vincent Hospital and Medical Center, Portland, Oregon; The Clinical Microbiology Institute, Tualatin, Oregon; the Department of Pathology, Kaiser Foundation Laboratories {Oregon Region}, Clackamas, Oregon; and the Antimicrobics and Resistance Mechanisms Branch, Centers for Disease Control, Atlanta, Georgia. Address reprint requests to: Peter C. Fuchs, M.D., Department of Pathology, St. Vincent Hospital and Medical Center, 9205 Southwest Barnes Road, Portland, OR 97225. Received April 26, 1983; revised and accepted August 5, 1983.
ElsevierScience Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017 © 1984
0732-8893/84/$03.00
56
P.C. Fuchs et al.
tococcus pyogenes; and 22 strains of Streptococcus agalactiae. The 310 Enterobacteriaceae isolates and the 246 other gram-negative bacteria are enumerated in Tables I and 2, respectively. These strains were collected from seven geographically separate institutions, as described p r e v i o u s l y (Fuchs et al., 1980). Susceptibility testing was performed by the broth m i c r o d i l u t i o n m e t h o d using m i c r o d i l u t i o n panels p r o v i d e d by Prepared Media Laboratory, Tualatin, OR. The antibiotics were d il u t e d in d i v a l e n t - c a t i o n - s u p p l e m e n t e d M u e l l e r - H i n t o n broth, as
TABLE 1. Susceptibility of 310 Enterobacteriaceae to T e m o c i l l i n and Five Other Antibiotics Organism (no. tested}
Antibiotic
MICso
MICgo
Escherichia coli (30)
TEM TIC CARl] CTX CTZ GENT
4.0 2.0 8.0 ~<0.06 ~<0.06 1.0
8.0 >256 >256 0.12 0.12 4.0
Klebsiella species (40)°
TEM TIC CARB CTX CTZ GENT
2.0 128 256 ~<0.06 ~<0.06 0.5
4.O >256 >256 ~<0.06 ~<0.06 0.5
Enterobacter species (60}b
TEM TIC CARB CTX CTZ GENT
2.0 4.0 4.0 0.12 ~<0.06 0.5
8.O >256 >256 8.O 8.0 1.0
Serratia marcescens (30)
TEM TIC CARB CTX CTZ GENT
8.O 4.0 8.O O.25 0.12 1.0
64 32 128 2.o O.5 1.0
Citrobacter diversus (20)
TEM TIC CARB CTX CTZ GENT
2.0 128 8.0 ~<0.06 ~<0.06 0.5
4.0 >256 16 0.12 0.12 1.0
Citrobacter freundii (21)
TEM TIC CARB CTX CTZ
2.0 2.0 4.0 0.12 0.12
8.0 >256 >256 0.25 0.5
GENT
0.5
1.0
In Vitro Activity of T e m o c i l l i n
57
TABLE 1 (continued) Organism (no. tested)
Antibiotic
MICso
MICgo
Morganel|a morganii (19)
TEM TIC CARl] CTX CTZ GENT
2.0 4.0 1.0 0.12 0.25 1.0
4.0 64 16 8.0 8.0 2.0
Proteus mirabilis (30)
TEM TIC CARB CTX CTZ GENT
1.0 ~0.5 ~0.5 ~0.06 ~0.06 1.0
2.0 ~0.5 ~0.5 ~0.06 ~0.06 2.0
Proteus vulgaris (20)
TEM TIC CARl] CTX CTZ GENT
2.0 4.0 4.0 ~0.06 ~0.06 1.0
4.0 64 64 0.12 ~<0.06 2.0
Providencia species (40)c
TEM TIC CARB CTX CTZ GENT
1.0 1.0 2.0 ~0.06 ~<0.06 4.0
2.0 >256 >256 1.0 ~0.06 32
Abbreviations: MIC = minimum inhibitory concentration; TEM = temocillin; TIC = ticarcillin; CARB = carbenicillin; CTX = cefotaxime;CTZ = ceftizoxime;GENT = gentamicin. "Includes K. pneumoniae (30) and K. oxytoca (10). bIncludes E. aerogenes (20), E. agglomerans (20), and E. cloacae (20). ~Includes P. rettgeri (20) and P. stuartii (20).
r e c o m m e n d e d by the National Committee for Clinical Laboratory Standards (NCCLS, 1983). For testing S. pyogenes, S. pneumoniae, H. influenzae, and Neisseria meningitidis, the broth was further s u p p l e m e n t e d with 5% Fildes peptic digest of horse blood. Testing was performed by the procedures r e c o m m e n d e d by the NCCLS (1983) and d e s c r i b e d p r e v i o u s l y (Ayers et al., 1982). These tests were c o n d u c t e d in two i n d e p e n d e n t laboratories (Centers for Disease Control, Atlanta, and Clinical Microbiology Institute, Tualatin), with at least 10% of isolates being tested at both laboratories as a control measure. Susceptibility testing of N. gonorrhoeae was performed at the Centers for Disease Control by agar dilution using proteose p e p t o n e no. 3 s u p p l e m e n t e d w i t h 1% hemoglobin and 1% Kellogg's s u p p l e m e n t (Baker et al., 1980). Determination of m i n i m a l bactericidal activity was performed by subculturing 5 p.l from each well (original concentration, 5 x 55 colony-forming units (CFU)/ml) of the microtiter m i n i m u m inhibitory concentration (MIC) tray onto a blood agar plate with a m u l t i p l e i n o c u l u m replicator. Endpoints were interpreted at 24 hr as the lowest concentration y i e l d i n g no more than 0.1% survivors (two colonies or fewer). Tests for B-lactamase stability and inhibitory activity of temocillin and comparison drugs were performed by m e t h o d s described previously (Ayers et al., 1982).
58
P.C. F u c h s e t al.
T A B L E Z. S u s c e p t i b i l i t y o f 2 4 6 N o n - E n t e r o b a c t e r i a c e a e to T e m o c i l l i n a n d F i v e O t h e r A n t i b i o t i c s O r g a n i s m (no. tested)
Acinetobacter s p e c i e s (20) a
Antibiotic TEM
Gram-Negative
MICso 64
Bacteria
MIC9o 128
TIC CARB CTX CTZ GENT
4.0 4.0 8.0 4.0 0.5
8.0 8.O 16 8.0 1.0
Pseudomonas aeruginosa (25)
TEM TIC CARB CTX CTZ GENT
>256 8.0 16 8.0 16 4.0
>256 16 32 32 64 8.0
P s e u d o m o n a s s p e c i e s (54) b
TEM TIC CARB CTX CTZ GENT
128 64 64 8.0 8.0 1.0
128 >256 >256 64 64 64
Haemophilus influenzae ~ - L a c t a m a s e n e g a t i v e (30)
TEM TIC CARB CTX CTZ GENT
0.25 0.5 0.5 O.06 0.06 1.0
0.5 0.5 0.5 0.06 O.06 2.0
~ - L a c t a m a s e p o s i t i v e (31)
TEM TIC CARB CTX CTZ GENT
0.12 2.0 2.0 0.06 0.06 2.o
0.25 16 8.0 0.06 0.06 2.o
TEM TIC CARB CTX CTZ GENT
0.12 O.5 0.5 0.06 0.06 4.0
0.12 0.5 0.5 O.06 0.06 8.0
~ - L a c t a m a s e n e g a t i v e (31)
TEM TIC
0.25 0.5
1.0 O.5
~ - L a c t a m a s e p o s i t i v e (30)
TF..M TIC
0.25 8.0
0.5 32
Neisseria meningitidis (25)
Neisseria gonorrhoeae
Abbreviations: MIC = m i n i m u m inhibitory concentration; TEM = temocillin; TIC = ticatcillin; CARl] = catbenicil[in; CTX = cefotaxime; CTZ = ceftizoxime; GENT = gentamicin. "Includes A. ca|coaceticus vat. anitratus (15) and vat. lwoffii (5). ~Includes P. acidovomns (5), P. cepacia (10), P. fluorescens (11), P. maltophilia (10), P. putida (8), and P. stutzeri (10].
In Vitro Activity of Temocillin
59
All gram-positive cocci tested were resistant to temocillin. The MICso and MICg0 (MICs at which 50% and 90% of strains are inhibited, respectively) for S. pyogenes were 64 p.g/ml, but for all other species were 128 vLg/mlor more. Temocillin exhibited good activity against Enterobacteriaceae (Table 1), with MICs generally within one dilution interval of those of ticarcillin. The major exception to this generalization involved Klebsiella pneumoniae and Citrobacter diversus, for which temocillin showed 64-fold greater activity than ticarcillin. The low MICro demonstrates that temocillin is active against many ticarcillin-resistant strains. Temocillin demonstrated no appreciable activity against Acinetobacter species or Pseudomonas species and, in fact, was often much less active than ticarcillin (Table 2). All five of the 13-1actam antibiotics tested were exceptionally active against 13lactamase-negative strains of H. influenzae, N. gonorrhoeae, and N. meningitidis, with an MICg0 of 0.5 ~g/ml or less. Whereas the MICs of ticarcillin and carbenicillin were significantly higher against [3-1actamase-producing strains, those of temocillin, cefotaxime, and ceftizoxime were unaffected (Table 2). Despite the variety of methods and test media used in various studies [Bolivar et ai., 1982; Greenwood et al., 1982; Jules and Neu, 1982; Slocombe et al., 1981; Van Landuyt et al., 1982; Verbist, 1982; Yogev et al., 1983}, the inhibitory activity of temocillin against the different species tested is remarkably comparable between the studies and with the current report (summarized in Table 3). In fact, the small differences observed (rarely more than one two-fold dilution step of the present study's endpoints) are more readily explained on the basis of differences between strains than between methodology or media. Temocillin demonstrated good bactericidal activity against Enterobacteriaceae, with minimal bactericidal concentrations (MBCs) averaging less than one dilution interval greater than the MICs (Table 4). The nine strains with MBCs three or more two-fold concentrations higher than the MICs included Enterobacter cloacae (4), Escherichia coli (2), K. pneumoniae (1), Morganella morganii (1), and Serratia marcescens (1). There was little inoculum effect (mean, 0.5 dilution interval) between concentrations of 103 and lO s CFU/ml. On the other hand, there was a significant reduction in inhibitory activity when the inoculum concentration of temocillin was increased from l0 s to 107 CFU/ml (mean, 3.5 dilution intervals of on-scale values}---quite comparable to the effect seen with ticarcillin (Table 4). Temocillin was quite stable to 13-1actamase hydrolysis (Table 5) by the commonly encountered enzyme types. In hydrolysis studies, compared to three frequently used labile substrates (benzyl penicillin, cephaloridine, and nitrocefin), the relative hydrolysis rate of temocillin was lower than that of the older cephalosporins, carboxypenicillins such as ticarcillin, and dicloxacillin. Trace temocillin hydrolysis was detected for E. cloacae P99 and E. coli TEM-2 enzymes, a feature not appreciated in earlier studies 0ules and Neu, 1982; Slocombe et al., 1981}. Only pseudomonad PSE2 and PSE-3 [3-1actamases have been shown to slowly break down temocillin (Jules and Neu, 1982}. ]ules and Neu (1982} reported significant inhibition of [3-1actamase hydrolysis of Richmond-Sykes type I enzymes by temocillin. Data in Table 6 confirm this finding with an E. cloacae P99 type I strain. However, the earlier report of inhibition of TEM1 (type III) 13-1actamase was not substantiated using the chromogenic enzyme-labile substrate PADAC. More studies concerning the enzyme-inhibiting qualities of temocillin are required. Temocillin exhibited good activity against Enterobacteriaceae and excellent activity against H. influenzae and pathogenic Neisseria, although in neither case did the MICs rival those of the third-generation cephalosporins. However, in view of its 13-1actamase stability, high achievable serum levels (Lagast and Klastersky, 1982;
60
P.C. F u c h s et al.
TABLE 3. C o m p a r i s o n of MICs0 a n d MICQo of T e m o c i l l i n i n P r e s e n t S t u d y w i t h T h o s e in Seven Previous Reports MICso MHB microdilution
Agar dilution
Organism
Escherichia coli Klebsiella species Enterobacter species Serratia marcescens Citrobacter species Morganella morganii Proteus mirabilis Proteus vulgaris Providencia species Haemophilus influenzae Unspecified t3-Lactamase negative ~-lactamase positive
BA base (Slocombe et al.) 5.0 2.5 2.5 12.5 2.5 2.5 2.5 2.5
MHA Oxoid DST (Verbist)
Jules & Neu
Van Landuyt et al.
4.0 2.0 2.0 16 4.0 2.0 1.0 2.0 1.0
4.0 4.0
8.0 4.0 4.0
0.2
32 4.0 2.0 8.0 2.0 1.0
Yogev et al.
4.0 4.0
Bolivar et al.
Present study
3.12 3.12 6.25 25 3.12
4.0 2.0 2.0 8.0 2.0 2.0 1.0 2.0 1.0
1.56 1.56 2.0
0.25 0.5
0.5
0.5
0.25
0.5
0.5
0.2
0.12
0.03
0.25
0.1
0.5
Neisseria gonorrhoeae Unspecified 13-Lactamase negative I]-Lactamase positive
Neisseria meningitidis
0.05
0.2
1.0
0.06
0.12
Abbreviations: MIC = minimum inhibitory concentration; BA = blood a8ar; MHA = Mueller-Hinton agar; MHB = Mueller-Hinton broth; DST : DST agar.
S l o c o m b e et al., 1981), a n d v e r y l o n g h a l f - l i f e of 5 h r ( S l o c o m b e et al., 1981), t e m o c i l l i n m a y b e w o r t h y of s e l e c t e d c l i n i c a l i n v e s t i g a t i o n s .
REFERENCES Ayers LW, Jones RN, Barry AL, Thornsberry C, Fuchs PC, Gavan TL, Gerlach EH, Sommers HM (1982) Cefotetan, a n e w cephamycin: comparison of in vitro antimicrobial activity with other cephems, beta-lactamase stability, and preliminary recommendations for disk diffusion testing. Antimicrob Agents Chemother 22:859. Baker CN, Thornsberry C, Jones RN (1980) In vitro antimicrobial activity of cefoperazone, cefotaxime, moxalactam (LY 127935), azlocillin, mezlocillin, and other beta-lactam antibiotics against N. gonorrhoeae and H. influenzae, including beta-lactamase producing strains. Antimicrob Agents Chemother 17:757. Bolivar R, Weaver SS, Bodey GP (1982) Comparative in vitro study of temocillin (BRL 17421), a new penicillin. Antimicrob Agents Chemother 21:641.
In Vitro Activity of Temocillin
TABLE 3
61
(continued) MIC~ MHB microdilution
Agar dilution BA base (Slocombe etal.)
MHA
Oxoid DST Greenwood etal.
12.5 5.0 5.0 25 5.0
8.0 8.0
2.5 2.5 5.0
4.0
Verbist 8.0 4.0 4.0 64 4.0 2.0 2.0 2.0 2.0
Van Lunduyt et al.
Bolivar etal.
8.0 16
12.5 8.0 8.0
8.0 12.5 12.5 50 12.5
Present study
4.0
4.0 8.0 64 8.0 4.0 2.0 4.0 2.0
0.5
0.5
0.5
0.5
1.0
0.25
0.5
1.0
Jules & Neu
256 16 4.0 8.0 4.0 4.0
8.0 8.0
1.56 1.56
0.5
1.0
2.0 1.0 0.5 0.12
0.12
Fuchs PC, Barry AL, Thornsberry C, Jones RN, Gavan TL, Gerlach EH, Sommers HM (1980) Cefotaxime: in vitro activity and tentative interpretive standards for disc susceptibility testing. Antimicrob Agents Chemother 18:88. Greenwood D, Cowlishaw A, Eley A (1982) In vitro activity of temocillin, a new beta-lactamasestable penicillin active against Enterobacteriaceae. Antimicrob Agents Chemother 22:198. Jones RN, Wilson HW (1983) Antimicrobial activity, beta-lactamase stability and beta-lactamase inhibition of cefotetan and other 7-alpha-methoxy beta-lactarn antimicrobials. Diegn Microbiol Infect Dis 1:71. Jules K, Neu HC (1982) Antibacterial activity and beta-lactamase stability of temocillin. Antimicrob Agents Chemother 22:453. Lagast H, Klastersky J (1982) Comparative in vitro activities of temocillin and cefazolin against Klebsiella pneumoniee. Antimicrob Agents Chemother 22:330. National Committee for Clinical Laboratory Standards (1983) Standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Tentative standard MTT. Villanova, PA: National Committee for Clinical Laboratory Standards.
T A B L E 4. B a c t e r i c i d a l A c t i v i t y a n d Effect of I n o c u l u m C o n c e n t r a t i o n o n A c t i v i t y of T e m o c i l l i n a n d T i c a r c i l l i n A g a i n s t 72 I s o l a t e s of E n t e r o b a c t e r i a c e a e Q Dilution intervals above a n d b e l o w M I C with 5 × 10 s C F U / m l I n o c u l u m Drug
Temocillin
Ticarcillin
I n o c u l u m size (CFU/rnl}
Endpoint
10 s 107 5 × 10 s
MIC MIC MBC
10 s 107 5 × 105
MIC MIC MBC
Off scale
- 4
- 3
3b
- 2
- 1
0
+ 1
+ 2
+ 3
+ 4
10
40
43
4 1 26
3 8
4 3
10 3
3 1
79 6
4 22
7 7
4 4
1 3
11 4
73 8
53 3
6
8
38
> + 4
Off scale
46 52
Abbreviations: M I C = m i n i m u m inhibitory concentration; C F U = colony-forming units; M B C = m i n i m u m bactericidalconcentration. alncludes I0 strainseach of CitrobacterJreundii, Escherichio coil, Enterobocter oerogenes, Enterobacter cloacae, K/ebsiella pneumonioe, and Serratia marcescens and 12 strainsof the Proteus-Providencia group. bValues represent percentages of 72 isolatestested.
In V i t r o A c t i v i t y of T e m o c i l l i n
63
T A B L E 5. ~-Lactamase Hydrolysis Rates of Temocillin C o m p a r e d with Those of Five Other ~-Lactams Temocillin RHR° compared to
Organism, ~-lactamase Bacillus cereus, N T b Enterobacter cloacae, P99 Escherichia coli,TEM-I Escherichia coli,TEM-2 Klebsiella oxytoca, K1
RHR compared to nitrocefin
Penicillin
Cephaloridine
Temocillin
Ticarcillin
Dicloxacillin
0.1 13 0.1 0.3
0.I 2.3 0.1 2.6
0.1 1.4 0.I 0.5
292 0.2 27 13
5.0 0.5 0.I 0.8
0.1
0.1
0.1
15
3.1
"Relative hydrolysis rate compared to that of nitrocefm, expressed as a percentage of 100. ~-Lactamase hydrolysis was determined by the ultraviolet spectrophotometric method using 235-570 nm at 37~C. Reaction mixtures were at a volume with 1.0 ml of 0.5-1.0 x 10--4 M cephalosporin and 1.0 x 10~ M penicillin substrates in 0.05 M phosphate buffer, pH 7. bNot typed (commercial penicillinase from B. cereus}.
Plot P, Van Dyck E {1962} In vitro activity of BRL 17421 against Haemophilus influenzae, Neisseria gonorrhoeae, and Branhamella catarrhalis.Antimicrob Agents Chemother 21:166. Richmond MH, Sykes RB {1973) The beta-lactamasesof gram-negative bacteriaand theirpossible physiological role. Adv Microb Physiol 9:31. Slocombe B, Basket MJ, Bentley PH, Clayton JP, Cole M, Comber I(R, Dixon RA, Edmondson RA, Jackson D, Merrikin DJ, Sutherland R {1981} BRL 17421, a novel beta-lecternantibiotic, highly resistant to beta-lactamases, giving high and prolonged serum levels in humans. Antimicrob Agents Chemother 20:36. Van Landuyt H W , Pyckavet M, Lambert A, Boelaert J {1962} In vitroactivityof temocillin {BRL 17421}, a novel beta-lecternantibiotic.Antimicrob Agents Chemother 22:535. Verbist L {1982} In vitro activityof temocillin {BRL 17421}, a novel beta-lactamase-stablepenicillin.Antimicrob Agents Chemother 22:157. Yogev R, Glogowski W, Conner E {1983} Comparative in vitroand in vivo activityof temocillin {BRL 17421) and ampicillin against FIoemophilus in~luenzae type b. Antimicrob Agents Chemother 23:182.
TABLE 6, I n h i b i t i o n of ~ - L a c t a m a s e H y d r o l y s i s of P A D A C b y E q u i m o l a r C o n c e n t r a t i o n s of T e m o c i l l i n a n d C o m p a r i s o n ~)-Lactams ° PADAC RHRb combined with Organism, [3-1actamase
Temocillin
Enterobecter cloacae, P99 F,scherichio coli,TEM-1 Escherichia coil TEM-2 K/ebsiella oxytoca, K1 Bacillus cereus, N T ~
1 84 86 88 90
Ticarcillin 1 35 25 80 100
Cefotaxime
Clavulanate
1 100 100 95 100
90 2 1 1 88
"Reference ~-lactamase-labile substrate {PADAC)concentration = 0.5 x 10-4 M or 31.1 p.g/mlin 0.05 M phosphate buffer, pH 7, combined with inhibiting ~-Iactam. bRalative hydrolysis rate compared to that of PADAC and added ~-lactamase, expressed as a percentage of 100. ~)LactAmsRehydrolysis was determined by a centrifugal-fast analyzer using 570 nm at 37°C. Reaction mixtures contained four different concentrations of inhibitor drug ranging from 0.1 to 100% that of the PADAC substrate concentration. CNot typed (commercial penicillinase from B. cereus).
55
NOTES
Temocillin: In Vitro Activity Against 734 Selected Clinical Isolates, Including -Lactamase-Producing Strains Peter C. Fuchs, Arthur L. Barry, Ronald N. Jones, and Clyde Thornsberry
The in vitro activity of temocillin against 734 clinical isolates was tested by broth microdilution. Good activity was demonstrated against Bnterobacteriaceae and both ff.loctamase-positive and -negative strains of Haemophilus int]uenzae and Neisser/a gonorrhoeoe. There was little to no activity against gram-positive cocci and nonfermenting gram-negative bacilli. Bactericidal activity and effect of inocu/um size on temociliin activity were comparable to that of ticarcillin. Temocillin was stable to commonly encountered [Mactamases and significantly inhibited Richmond-Sykes type 1 enzymes of Enterobacter cloacae.
Temocillin (BRL 17421} is a new semisynthetic penicillin structurally similar to ticarcillin, but differing from ticarcillinby the introduction of a methoxy group at the 6-a position of the ~-lactam ring. This modification has rendered temocillin resistant to ~-lactamases, but less active against Pseudomonas species (Julesand Neu, 1982; Slocombe et al.,1981}. Good in vitro activity against a wide spectrum of other bacteria, including most strains of Enterobacteriaceae, Hoemophilus influenzae,and Neisserio gonorrhoeae, has been reported previously {Bolivar et al.,1982; Greenwood et al., 1982; Lagast and Klastersky, 1982; Plot and Van Dyck, 1982; Van Landuyt et al., 1982; Verbist, 1982; Yogev eta]., 1983}. The present study compares the in vitro activity of temocillin with that of related semisynthetic penicillins {ticarcillinand carbenicillin}, newer cephalosporins {cefotaxime and ceftizoxime), and gentamicin against a broad range of recent clinical isolates. A total of 734 clinical isolates was studied. The 178 gram-positive coccal isolates i n c l u d e d 60 strains of Staphylococcus aureus, 10 of w h i c h were methicillin resistant a n d 27 of w h i c h p r o d u c e d ~-lactamase, but were methicillin susceptible; 24 strains of S t a p h y l o c o c c u s epidermidis, 14 of w h i c h were p e n i c i l l i n resistant; 25 strains of Streptococcus faecolis; 26 strains of Streptococcus pneumonioe; 21 strains of Strep-
From the Department of Pathology, St. Vincent Hospital and Medical Center, Portland, Oregon; The Clinical Microbiology Institute, Tualatin, Oregon; the Department of Pathology, Kaiser Foundation Laboratories {Oregon Region}, Clackamas, Oregon; and the Antimicrobics and Resistance Mechanisms Branch, Centers for Disease Control, Atlanta, Georgia. Address reprint requests to: Peter C. Fuchs, M.D., Department of Pathology, St. Vincent Hospital and Medical Center, 9205 Southwest Barnes Road, Portland, OR 97225. Received April 26, 1983; revised and accepted August 5, 1983.
ElsevierScience Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017 © 1984
0732-8893/84/$03.00
56
P.C. Fuchs et al.
tococcus pyogenes; and 22 strains of Streptococcus agalactiae. The 310 Enterobacteriaceae isolates and the 246 other gram-negative bacteria are enumerated in Tables I and 2, respectively. These strains were collected from seven geographically separate institutions, as described p r e v i o u s l y (Fuchs et al., 1980). Susceptibility testing was performed by the broth m i c r o d i l u t i o n m e t h o d using m i c r o d i l u t i o n panels p r o v i d e d by Prepared Media Laboratory, Tualatin, OR. The antibiotics were d il u t e d in d i v a l e n t - c a t i o n - s u p p l e m e n t e d M u e l l e r - H i n t o n broth, as
TABLE 1. Susceptibility of 310 Enterobacteriaceae to T e m o c i l l i n and Five Other Antibiotics Organism (no. tested}
Antibiotic
MICso
MICgo
Escherichia coli (30)
TEM TIC CARl] CTX CTZ GENT
4.0 2.0 8.0 ~<0.06 ~<0.06 1.0
8.0 >256 >256 0.12 0.12 4.0
Klebsiella species (40)°
TEM TIC CARB CTX CTZ GENT
2.0 128 256 ~<0.06 ~<0.06 0.5
4.O >256 >256 ~<0.06 ~<0.06 0.5
Enterobacter species (60}b
TEM TIC CARB CTX CTZ GENT
2.0 4.0 4.0 0.12 ~<0.06 0.5
8.O >256 >256 8.O 8.0 1.0
Serratia marcescens (30)
TEM TIC CARB CTX CTZ GENT
8.O 4.0 8.O O.25 0.12 1.0
64 32 128 2.o O.5 1.0
Citrobacter diversus (20)
TEM TIC CARB CTX CTZ GENT
2.0 128 8.0 ~<0.06 ~<0.06 0.5
4.0 >256 16 0.12 0.12 1.0
Citrobacter freundii (21)
TEM TIC CARB CTX CTZ
2.0 2.0 4.0 0.12 0.12
8.0 >256 >256 0.25 0.5
GENT
0.5
1.0
In Vitro Activity of T e m o c i l l i n
57
TABLE 1 (continued) Organism (no. tested)
Antibiotic
MICso
MICgo
Morganel|a morganii (19)
TEM TIC CARl] CTX CTZ GENT
2.0 4.0 1.0 0.12 0.25 1.0
4.0 64 16 8.0 8.0 2.0
Proteus mirabilis (30)
TEM TIC CARB CTX CTZ GENT
1.0 ~0.5 ~0.5 ~0.06 ~0.06 1.0
2.0 ~0.5 ~0.5 ~0.06 ~0.06 2.0
Proteus vulgaris (20)
TEM TIC CARl] CTX CTZ GENT
2.0 4.0 4.0 ~0.06 ~0.06 1.0
4.0 64 64 0.12 ~<0.06 2.0
Providencia species (40)c
TEM TIC CARB CTX CTZ GENT
1.0 1.0 2.0 ~0.06 ~<0.06 4.0
2.0 >256 >256 1.0 ~0.06 32
Abbreviations: MIC = minimum inhibitory concentration; TEM = temocillin; TIC = ticarcillin; CARB = carbenicillin; CTX = cefotaxime;CTZ = ceftizoxime;GENT = gentamicin. "Includes K. pneumoniae (30) and K. oxytoca (10). bIncludes E. aerogenes (20), E. agglomerans (20), and E. cloacae (20). ~Includes P. rettgeri (20) and P. stuartii (20).
r e c o m m e n d e d by the National Committee for Clinical Laboratory Standards (NCCLS, 1983). For testing S. pyogenes, S. pneumoniae, H. influenzae, and Neisseria meningitidis, the broth was further s u p p l e m e n t e d with 5% Fildes peptic digest of horse blood. Testing was performed by the procedures r e c o m m e n d e d by the NCCLS (1983) and d e s c r i b e d p r e v i o u s l y (Ayers et al., 1982). These tests were c o n d u c t e d in two i n d e p e n d e n t laboratories (Centers for Disease Control, Atlanta, and Clinical Microbiology Institute, Tualatin), with at least 10% of isolates being tested at both laboratories as a control measure. Susceptibility testing of N. gonorrhoeae was performed at the Centers for Disease Control by agar dilution using proteose p e p t o n e no. 3 s u p p l e m e n t e d w i t h 1% hemoglobin and 1% Kellogg's s u p p l e m e n t (Baker et al., 1980). Determination of m i n i m a l bactericidal activity was performed by subculturing 5 p.l from each well (original concentration, 5 x 55 colony-forming units (CFU)/ml) of the microtiter m i n i m u m inhibitory concentration (MIC) tray onto a blood agar plate with a m u l t i p l e i n o c u l u m replicator. Endpoints were interpreted at 24 hr as the lowest concentration y i e l d i n g no more than 0.1% survivors (two colonies or fewer). Tests for B-lactamase stability and inhibitory activity of temocillin and comparison drugs were performed by m e t h o d s described previously (Ayers et al., 1982).
58
P.C. F u c h s e t al.
T A B L E Z. S u s c e p t i b i l i t y o f 2 4 6 N o n - E n t e r o b a c t e r i a c e a e to T e m o c i l l i n a n d F i v e O t h e r A n t i b i o t i c s O r g a n i s m (no. tested)
Acinetobacter s p e c i e s (20) a
Antibiotic TEM
Gram-Negative
MICso 64
Bacteria
MIC9o 128
TIC CARB CTX CTZ GENT
4.0 4.0 8.0 4.0 0.5
8.0 8.O 16 8.0 1.0
Pseudomonas aeruginosa (25)
TEM TIC CARB CTX CTZ GENT
>256 8.0 16 8.0 16 4.0
>256 16 32 32 64 8.0
P s e u d o m o n a s s p e c i e s (54) b
TEM TIC CARB CTX CTZ GENT
128 64 64 8.0 8.0 1.0
128 >256 >256 64 64 64
Haemophilus influenzae ~ - L a c t a m a s e n e g a t i v e (30)
TEM TIC CARB CTX CTZ GENT
0.25 0.5 0.5 O.06 0.06 1.0
0.5 0.5 0.5 0.06 O.06 2.0
~ - L a c t a m a s e p o s i t i v e (31)
TEM TIC CARB CTX CTZ GENT
0.12 2.0 2.0 0.06 0.06 2.o
0.25 16 8.0 0.06 0.06 2.o
TEM TIC CARB CTX CTZ GENT
0.12 O.5 0.5 0.06 0.06 4.0
0.12 0.5 0.5 O.06 0.06 8.0
~ - L a c t a m a s e n e g a t i v e (31)
TEM TIC
0.25 0.5
1.0 O.5
~ - L a c t a m a s e p o s i t i v e (30)
TF..M TIC
0.25 8.0
0.5 32
Neisseria meningitidis (25)
Neisseria gonorrhoeae
Abbreviations: MIC = m i n i m u m inhibitory concentration; TEM = temocillin; TIC = ticatcillin; CARl] = catbenicil[in; CTX = cefotaxime; CTZ = ceftizoxime; GENT = gentamicin. "Includes A. ca|coaceticus vat. anitratus (15) and vat. lwoffii (5). ~Includes P. acidovomns (5), P. cepacia (10), P. fluorescens (11), P. maltophilia (10), P. putida (8), and P. stutzeri (10].
In Vitro Activity of Temocillin
59
All gram-positive cocci tested were resistant to temocillin. The MICso and MICg0 (MICs at which 50% and 90% of strains are inhibited, respectively) for S. pyogenes were 64 p.g/ml, but for all other species were 128 vLg/mlor more. Temocillin exhibited good activity against Enterobacteriaceae (Table 1), with MICs generally within one dilution interval of those of ticarcillin. The major exception to this generalization involved Klebsiella pneumoniae and Citrobacter diversus, for which temocillin showed 64-fold greater activity than ticarcillin. The low MICro demonstrates that temocillin is active against many ticarcillin-resistant strains. Temocillin demonstrated no appreciable activity against Acinetobacter species or Pseudomonas species and, in fact, was often much less active than ticarcillin (Table 2). All five of the 13-1actam antibiotics tested were exceptionally active against 13lactamase-negative strains of H. influenzae, N. gonorrhoeae, and N. meningitidis, with an MICg0 of 0.5 ~g/ml or less. Whereas the MICs of ticarcillin and carbenicillin were significantly higher against [3-1actamase-producing strains, those of temocillin, cefotaxime, and ceftizoxime were unaffected (Table 2). Despite the variety of methods and test media used in various studies [Bolivar et ai., 1982; Greenwood et al., 1982; Jules and Neu, 1982; Slocombe et al., 1981; Van Landuyt et al., 1982; Verbist, 1982; Yogev et al., 1983}, the inhibitory activity of temocillin against the different species tested is remarkably comparable between the studies and with the current report (summarized in Table 3). In fact, the small differences observed (rarely more than one two-fold dilution step of the present study's endpoints) are more readily explained on the basis of differences between strains than between methodology or media. Temocillin demonstrated good bactericidal activity against Enterobacteriaceae, with minimal bactericidal concentrations (MBCs) averaging less than one dilution interval greater than the MICs (Table 4). The nine strains with MBCs three or more two-fold concentrations higher than the MICs included Enterobacter cloacae (4), Escherichia coli (2), K. pneumoniae (1), Morganella morganii (1), and Serratia marcescens (1). There was little inoculum effect (mean, 0.5 dilution interval) between concentrations of 103 and lO s CFU/ml. On the other hand, there was a significant reduction in inhibitory activity when the inoculum concentration of temocillin was increased from l0 s to 107 CFU/ml (mean, 3.5 dilution intervals of on-scale values}---quite comparable to the effect seen with ticarcillin (Table 4). Temocillin was quite stable to 13-1actamase hydrolysis (Table 5) by the commonly encountered enzyme types. In hydrolysis studies, compared to three frequently used labile substrates (benzyl penicillin, cephaloridine, and nitrocefin), the relative hydrolysis rate of temocillin was lower than that of the older cephalosporins, carboxypenicillins such as ticarcillin, and dicloxacillin. Trace temocillin hydrolysis was detected for E. cloacae P99 and E. coli TEM-2 enzymes, a feature not appreciated in earlier studies 0ules and Neu, 1982; Slocombe et al., 1981}. Only pseudomonad PSE2 and PSE-3 [3-1actamases have been shown to slowly break down temocillin (Jules and Neu, 1982}. ]ules and Neu (1982} reported significant inhibition of [3-1actamase hydrolysis of Richmond-Sykes type I enzymes by temocillin. Data in Table 6 confirm this finding with an E. cloacae P99 type I strain. However, the earlier report of inhibition of TEM1 (type III) 13-1actamase was not substantiated using the chromogenic enzyme-labile substrate PADAC. More studies concerning the enzyme-inhibiting qualities of temocillin are required. Temocillin exhibited good activity against Enterobacteriaceae and excellent activity against H. influenzae and pathogenic Neisseria, although in neither case did the MICs rival those of the third-generation cephalosporins. However, in view of its 13-1actamase stability, high achievable serum levels (Lagast and Klastersky, 1982;
60
P.C. F u c h s et al.
TABLE 3. C o m p a r i s o n of MICs0 a n d MICQo of T e m o c i l l i n i n P r e s e n t S t u d y w i t h T h o s e in Seven Previous Reports MICso MHB microdilution
Agar dilution
Organism
Escherichia coli Klebsiella species Enterobacter species Serratia marcescens Citrobacter species Morganella morganii Proteus mirabilis Proteus vulgaris Providencia species Haemophilus influenzae Unspecified t3-Lactamase negative ~-lactamase positive
BA base (Slocombe et al.) 5.0 2.5 2.5 12.5 2.5 2.5 2.5 2.5
MHA Oxoid DST (Verbist)
Jules & Neu
Van Landuyt et al.
4.0 2.0 2.0 16 4.0 2.0 1.0 2.0 1.0
4.0 4.0
8.0 4.0 4.0
0.2
32 4.0 2.0 8.0 2.0 1.0
Yogev et al.
4.0 4.0
Bolivar et al.
Present study
3.12 3.12 6.25 25 3.12
4.0 2.0 2.0 8.0 2.0 2.0 1.0 2.0 1.0
1.56 1.56 2.0
0.25 0.5
0.5
0.5
0.25
0.5
0.5
0.2
0.12
0.03
0.25
0.1
0.5
Neisseria gonorrhoeae Unspecified 13-Lactamase negative I]-Lactamase positive
Neisseria meningitidis
0.05
0.2
1.0
0.06
0.12
Abbreviations: MIC = minimum inhibitory concentration; BA = blood a8ar; MHA = Mueller-Hinton agar; MHB = Mueller-Hinton broth; DST : DST agar.
S l o c o m b e et al., 1981), a n d v e r y l o n g h a l f - l i f e of 5 h r ( S l o c o m b e et al., 1981), t e m o c i l l i n m a y b e w o r t h y of s e l e c t e d c l i n i c a l i n v e s t i g a t i o n s .
REFERENCES Ayers LW, Jones RN, Barry AL, Thornsberry C, Fuchs PC, Gavan TL, Gerlach EH, Sommers HM (1982) Cefotetan, a n e w cephamycin: comparison of in vitro antimicrobial activity with other cephems, beta-lactamase stability, and preliminary recommendations for disk diffusion testing. Antimicrob Agents Chemother 22:859. Baker CN, Thornsberry C, Jones RN (1980) In vitro antimicrobial activity of cefoperazone, cefotaxime, moxalactam (LY 127935), azlocillin, mezlocillin, and other beta-lactam antibiotics against N. gonorrhoeae and H. influenzae, including beta-lactamase producing strains. Antimicrob Agents Chemother 17:757. Bolivar R, Weaver SS, Bodey GP (1982) Comparative in vitro study of temocillin (BRL 17421), a new penicillin. Antimicrob Agents Chemother 21:641.
In Vitro Activity of Temocillin
TABLE 3
61
(continued) MIC~ MHB microdilution
Agar dilution BA base (Slocombe etal.)
MHA
Oxoid DST Greenwood etal.
12.5 5.0 5.0 25 5.0
8.0 8.0
2.5 2.5 5.0
4.0
Verbist 8.0 4.0 4.0 64 4.0 2.0 2.0 2.0 2.0
Van Lunduyt et al.
Bolivar etal.
8.0 16
12.5 8.0 8.0
8.0 12.5 12.5 50 12.5
Present study
4.0
4.0 8.0 64 8.0 4.0 2.0 4.0 2.0
0.5
0.5
0.5
0.5
1.0
0.25
0.5
1.0
Jules & Neu
256 16 4.0 8.0 4.0 4.0
8.0 8.0
1.56 1.56
0.5
1.0
2.0 1.0 0.5 0.12
0.12
Fuchs PC, Barry AL, Thornsberry C, Jones RN, Gavan TL, Gerlach EH, Sommers HM (1980) Cefotaxime: in vitro activity and tentative interpretive standards for disc susceptibility testing. Antimicrob Agents Chemother 18:88. Greenwood D, Cowlishaw A, Eley A (1982) In vitro activity of temocillin, a new beta-lactamasestable penicillin active against Enterobacteriaceae. Antimicrob Agents Chemother 22:198. Jones RN, Wilson HW (1983) Antimicrobial activity, beta-lactamase stability and beta-lactamase inhibition of cefotetan and other 7-alpha-methoxy beta-lactarn antimicrobials. Diegn Microbiol Infect Dis 1:71. Jules K, Neu HC (1982) Antibacterial activity and beta-lactamase stability of temocillin. Antimicrob Agents Chemother 22:453. Lagast H, Klastersky J (1982) Comparative in vitro activities of temocillin and cefazolin against Klebsiella pneumoniee. Antimicrob Agents Chemother 22:330. National Committee for Clinical Laboratory Standards (1983) Standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Tentative standard MTT. Villanova, PA: National Committee for Clinical Laboratory Standards.
T A B L E 4. B a c t e r i c i d a l A c t i v i t y a n d Effect of I n o c u l u m C o n c e n t r a t i o n o n A c t i v i t y of T e m o c i l l i n a n d T i c a r c i l l i n A g a i n s t 72 I s o l a t e s of E n t e r o b a c t e r i a c e a e Q Dilution intervals above a n d b e l o w M I C with 5 × 10 s C F U / m l I n o c u l u m Drug
Temocillin
Ticarcillin
I n o c u l u m size (CFU/rnl}
Endpoint
10 s 107 5 × 10 s
MIC MIC MBC
10 s 107 5 × 105
MIC MIC MBC
Off scale
- 4
- 3
3b
- 2
- 1
0
+ 1
+ 2
+ 3
+ 4
10
40
43
4 1 26
3 8
4 3
10 3
3 1
79 6
4 22
7 7
4 4
1 3
11 4
73 8
53 3
6
8
38
> + 4
Off scale
46 52
Abbreviations: M I C = m i n i m u m inhibitory concentration; C F U = colony-forming units; M B C = m i n i m u m bactericidalconcentration. alncludes I0 strainseach of CitrobacterJreundii, Escherichio coil, Enterobocter oerogenes, Enterobacter cloacae, K/ebsiella pneumonioe, and Serratia marcescens and 12 strainsof the Proteus-Providencia group. bValues represent percentages of 72 isolatestested.
In V i t r o A c t i v i t y of T e m o c i l l i n
63
T A B L E 5. ~-Lactamase Hydrolysis Rates of Temocillin C o m p a r e d with Those of Five Other ~-Lactams Temocillin RHR° compared to
Organism, ~-lactamase Bacillus cereus, N T b Enterobacter cloacae, P99 Escherichia coli,TEM-I Escherichia coli,TEM-2 Klebsiella oxytoca, K1
RHR compared to nitrocefin
Penicillin
Cephaloridine
Temocillin
Ticarcillin
Dicloxacillin
0.1 13 0.1 0.3
0.I 2.3 0.1 2.6
0.1 1.4 0.I 0.5
292 0.2 27 13
5.0 0.5 0.I 0.8
0.1
0.1
0.1
15
3.1
"Relative hydrolysis rate compared to that of nitrocefm, expressed as a percentage of 100. ~-Lactamase hydrolysis was determined by the ultraviolet spectrophotometric method using 235-570 nm at 37~C. Reaction mixtures were at a volume with 1.0 ml of 0.5-1.0 x 10--4 M cephalosporin and 1.0 x 10~ M penicillin substrates in 0.05 M phosphate buffer, pH 7. bNot typed (commercial penicillinase from B. cereus}.
Plot P, Van Dyck E {1962} In vitro activity of BRL 17421 against Haemophilus influenzae, Neisseria gonorrhoeae, and Branhamella catarrhalis.Antimicrob Agents Chemother 21:166. Richmond MH, Sykes RB {1973) The beta-lactamasesof gram-negative bacteriaand theirpossible physiological role. Adv Microb Physiol 9:31. Slocombe B, Basket MJ, Bentley PH, Clayton JP, Cole M, Comber I(R, Dixon RA, Edmondson RA, Jackson D, Merrikin DJ, Sutherland R {1981} BRL 17421, a novel beta-lecternantibiotic, highly resistant to beta-lactamases, giving high and prolonged serum levels in humans. Antimicrob Agents Chemother 20:36. Van Landuyt H W , Pyckavet M, Lambert A, Boelaert J {1962} In vitroactivityof temocillin {BRL 17421}, a novel beta-lecternantibiotic.Antimicrob Agents Chemother 22:535. Verbist L {1982} In vitro activityof temocillin {BRL 17421}, a novel beta-lactamase-stablepenicillin.Antimicrob Agents Chemother 22:157. Yogev R, Glogowski W, Conner E {1983} Comparative in vitroand in vivo activityof temocillin {BRL 17421) and ampicillin against FIoemophilus in~luenzae type b. Antimicrob Agents Chemother 23:182.
TABLE 6, I n h i b i t i o n of ~ - L a c t a m a s e H y d r o l y s i s of P A D A C b y E q u i m o l a r C o n c e n t r a t i o n s of T e m o c i l l i n a n d C o m p a r i s o n ~)-Lactams ° PADAC RHRb combined with Organism, [3-1actamase
Temocillin
Enterobecter cloacae, P99 F,scherichio coli,TEM-1 Escherichia coil TEM-2 K/ebsiella oxytoca, K1 Bacillus cereus, N T ~
1 84 86 88 90
Ticarcillin 1 35 25 80 100
Cefotaxime
Clavulanate
1 100 100 95 100
90 2 1 1 88
"Reference ~-lactamase-labile substrate {PADAC)concentration = 0.5 x 10-4 M or 31.1 p.g/mlin 0.05 M phosphate buffer, pH 7, combined with inhibiting ~-Iactam. bRalative hydrolysis rate compared to that of PADAC and added ~-lactamase, expressed as a percentage of 100. ~)LactAmsRehydrolysis was determined by a centrifugal-fast analyzer using 570 nm at 37°C. Reaction mixtures contained four different concentrations of inhibitor drug ranging from 0.1 to 100% that of the PADAC substrate concentration. CNot typed (commercial penicillinase from B. cereus).