Journal of Antimicrobial Chemotherapy (1979) 5, 293-300
Comparative activity of two newer cephalosponns, cefoxitin, and cephalothin against selected Enterobacteriaceae and correlation with enzymatic resistance mechanisms
Department of Microbiology, Faculty of Pharmacy, University of Gent, Apotheekstraat 1, B-9000 Gent, Belgium
and H. W. Van Landnyt,
Department of Microbiology, General Hospital St-Jan, Brugge, Belgium
A comparative in vitro investigation of three newer P-lactam antibiotics (cefamandole, cefuroxime and cefoxitin) against ampicillin and/or cephalothin resistant Enterobacteriaceae was performed by disc diffusion testing and by continuous turbidimetric monitoring. Cefamandole displayed good activity against cephalosporinase-producing organisms, with the exception of serratia and Proteus vulgaris. Production of broad spectrum enzymes generally resulted in rapid inactivation of cefamandole. In contrast, cefuroxime and cefoxitin demonstrated high resistance against most enterobacterial P-lactamases. Cefoxitin was the only antibiotic with considerable activity against serratia. The same compound was inactive against enterobacter in conventional tests, but nevertheless did demonstrate a transient inhibitory effect in the turbidimetric system. Introduction
The cephalosporins have a wide spectrum of activity against both Gram-positive and Gram-negative organisms. However, conventional cephalosporins (cephalothin, cephaloridine, cephalexin, cefazolin) have only minor activity against certain Enterobacteriaceae such as serratia, enterobacter, indole-positive proteus and providencia. This resistance is primarily due to hydrolytic attack by different P-lactamases (Sabath & Finland, 1967; Farrar & Krause, 1970). Recently a number of new p-lactam antibiotics have been introduced which show a markedly wider spectrum of activity against Gram-negative bacilli. The most promising of these compounds are cefamandole and cefuroxime, which are cephalosporins and cefoxitin which belongs to the cephamycins. The purpose of this study was to compare the in vitro activity of these newer antibiotics with respect to a number of selected ampicillin and/or cephalothin resistant 0305-7453/79/030293+08 ?01.00/0
293 © 1979 The British Society for Antimicrobial Chemotherapy
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A. Vuye, J. Pijck,
294
A. Vuye, J. Pijck and H. W. Van Landuyt
Enterobacteriaceae. In order to provide a rationale for understanding some of the differences in antimicrobial activity of the compounds, enzymatic resistance mechanisms were characterized in all tested organisms. Materials and methods Bacterial strains
Investigation of enzymatic resistance mechanisms Preparation of enzymes. Crude extracts were obtained by sonic disruption of cell suspensions, followed by removal of cell debris by centrifugation. Characterization of $-lactamases. Electrophoretic separations of the crude enzymes were performed at pH 8-5 by the starch-gel method of Smithies (1955). P-Lactamases were recorded as broad spectrum (BS) enzymes or cephalosporinases (Cs), by comparing the relative rates of hydrolysis of benzylpenicillin and cephaloridine at a substrate concentration of 5 mM. A modification of the micro-iodometric method ofNovick (1962) was used for determination of enzymatic activity with these compounds as substrate. Relative rates of enzymatic hydrolysis of the cephalosporins at a substrate concentration of 10~4 M were determined by the spectrophotometric method, described by Ross & O'Callaghan (1975). Transfer of R-plasmids. Organisms were mated with a rifampicin or nalidixic acid resistant substrain of E. coli K12. Transconjugants were selected on antibiotic containing selective media, purified, and tested for drug resistance and production of (i-lactamases. Resistance to ampicillin in the transconjugants obtained in this way, was primarily due to fl-lactamase production, and provided evidence of the extrachromosomal genetic basis for p-lactamase formation. When failure of conjugational transfer of the pMactamase was observed, the enzyme was scored as probably chromosomally mediated. However, to locate unequivocally a gene on the bacterial chromosome, more sophisticated methods are needed. Susceptibility tests Antibiotic susceptibility of all organisms tested was determined on Mueller-Hinton agar by the standard disc diffusion method of Bauer, Kirby, Sherris & Turck (1966). Organisms which gave a zone of inhibition of 18 mm diameter or greater, obtained with 0-6 cm discs containing 30 ug of the respective cephalosporins, were designated susceptible to these compounds. For ampicillin (10 ug) and carbenicillin (100 ug), 14 and 23 mm were respectively taken as the minimum inhibition zone diameters, representing a sensitive response.
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Most of the strains were recently isolated from patients in St-Jan's Hospital, Brugge. The selection of Escherichia coli and klebsiella strains took place on the basis of preliminary susceptibility tests with ampicillin and cephalothin. Only strains resistant to one or both of these compounds were further investigated. In general, the remaining species investigated (Enterobacter aerogenes, E. cloacae, Serratia marcescens, Proteus morganii, P. vulgaris, P. rettgeri and Providencia stuartii) were resistant to cephalothin. Consequently, all strains of these species were selected for further investigation.
Activity of cefamandole, cefaroxime, cefoxitin
295
Antibiotics Cephalothin and cefamandole were provided by Eli Lilly and Co., cefuroxime by Glaxo and cefoxitin by Merck, Sharp and Dohme. Results The profiles of the enzymes produced in different Gram-negative species are indicated in Table I, together with disc diffusion susceptibilities to 2 penicillins, 3 cephalosporins and cefoxitin. Characteristics of the enzymes are scored in Table n . Production of broad spectrum enzymes, which were either chromosomally or R factor-mediated was observed in all genera, except enterobacter. Among the klebsiella strains, one isolate was found that produced a rather exceptional enzyme (BS3) which could not be identified with one of the enzyme types, described by Richmond & Sykes (1973). This enzyme was a constitutive broad spectrum P-lactamase, with an electrophoretic mobility slightly lower than that of EUa (TEM) enzyme. It hydrolysed to a considerable degree all fJ-lactam antibiotics, with the exception of cefoxitin. Typical cephalosporinases, which could never be transferred by conjugation, were found in all genera except klebsiella. The Cs enzymes produced by E. coli differed from other cephalosporinases in that they were invariably constitutive. All strains of Proteus rettgeri were found to produce broad spectrum enzymes in addition to their speciesspecific cephalosporinases. Broad spectrum enzymes, whether chromosomally or R factor-mediated invariably caused resistance to both ampicillin and carbenicillin. Production of a cephalosporinase was generally indicative for high-degree resistance against cephalothin, regardless of the genus concerned. The newer compounds (cefamandole, cefuroxime and cefoxitin) were active on a great number of the ampicillin and/or cephalothin resistant isolates. In general, cefoxitin had the most reliable activity but it was ineffective against enterobacter, when susceptibility was determined with conventional methods. Cefoxitin furthermore proved to be the only fl-lactam antibiotic with reliable activity against Serratia marcescens. Rates of hydrolysis of the different cephalosporins [relative to hydrolysis of cephaloridine ( = 100)] are illustrated in Table EL Cefoxitin was clearly the most stable compound
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From each of the different enzymatic resistance groups observed, a limited number of strains were selected for further susceptibility testing by continuous turbidimetric monitoring. The turbidimetric system consisted of six stirred cultures in Mueller-Hinton broth, which were continuously monitored in a Bonet-Maury biophotometer (Jobin Yvon instruments). Antibiotics were added at the same point in the logarithmic phase, when the optical density was equivalent to a viable count of about 1 x 107 bacteria/ml. These conditions differ markedly from those of conventional susceptibility tests, but it may be argued that the denser population represents the numbers of bacteria in vivo more closely (Greenwood & O'Grady, 1973). The concentration of antibiotic was fixed at the cut-off point of 32 ug/ml, representing the maximum blood level concentration of most cephalosporins. The time elapsing between addition of antibiotic and lysis of the culture and the time before regrowth occurred were both noted. In this way information can be obtained on both the intrinsic activity of the compounds and their susceptibility to enzymatic inactivation (Greenwood, 1977).
A- Vuye, J. Pijck and H. W. Van Landnyt
296
Table L Distribution of p-lactamases among ampicillin and/or cephalothin resistant Enterobacteriaceae with indication of susceptibility to different 3-lactam antibiotics
Organ isn:i (total no. of strains) E. coli
(39)
P-lactamases produced BS*
Cst
Klebsiella
(22)
P. morganii
(13)
P. vulgaris
(10)
P. rettgeri Prov. stuartii
(7) (9)
No. of strains resistant to:
enzyme(s) 29 7 3 10 11 1 3 11 10 8 2 11 1 9 7 6 3
APt CB CF CA CU CO 29 29 19 7 0 7 3 3 3 10 10 9 11 11 3 1 1 1 0 0 3 1 11 3 10 9 10 7 0 8 2 2 2 6 0 11 1 1 1 9 1 9 7 7 6 6 6 4 2 0 3
2 0 4 1 1 1 2 2 3 0 2 0 1 0 1 1 0 1 1 2 0 1 1 10 1 10 10 8 7 1 1 2 0 1 2 0 1 0 1 8 6 1 1 0 3 0 0 0 0 0 0
* BS, broad spectrum enzyme t Cs, cephalosporinase. \ AP, ampicillin. CB, carbenicillin; CF, cephalothin; CA, cefamandole; CU, cefuroxinie; CO, cefoxitin.
since none of the investigated enzymes was able to hydrolyze this antibiotic. Cefamandole was found to be rapidly hydrolyzed by broad spectrum enzymes, but stable against cephalosporinases, with the exception of those from Proteus vulgaris. Figure 1 illustrates graphically the time between the addition of antibiotic and the recommencement of growth after lysis as shown by turbidimetric measurements. Trie strains selected for the figure were representative for the average response of isolates belonging to the same enzymatic resistance class. Cefamandole, like cephalothin, showed only a transient inhibitory effect against most organisms with R factor-mediated enzymes. With several strains (e.g. most strains of serratia) cefamandole and cefuroxime displayed only incomplete lytic effect, indicating lack of intrinsic activity (turbidity fell to an intermediate level, which was maintained during variable time periods). Biophotometer tracings obtained with different enterobacter strains showed that cefamandole and cefuroxime suppressed growth only slightly longer than cefoxitin. The extent of lysis before regrowth occurred was similar for the three compounds. Thus, lack of susceptibility of enterobacter to cefoxitin demonstrated in conventional susceptibility tests could not be corroborated by turbidimetric monitoring under the defined conditions. The mechanism of cefoxitin resistance in enterobacter clearly needs further investigation. Intrinsic activity as shown by rapidity and extent of lysis was generally highest for cefoxitin. Cefuroxime as a rule was less intrinsically active than cefamandole.
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E. aerogenes (3) E. cloacae (11) S. marcescens (18)
BS + Cs BS! BS, BS. Cs Cs BS + Cs Cs BS + Cs Cs BS + Cs Cs BS + Cs BS Cs
No. of strains with inHipfltcH
9/53 23/77 29/85 49 65 9/78 10 23 34 23/53 23/80 33 9/57 64 19B 23/41 101
R C C R C C C
NI NI
NI NI I I I NI I NI I NI NI I
BS, BS.
BS, BS
Cs
BS BS
Cs
BS
Cs
Cs BS
Cs Cs
C
R R
C
R
C
R
C
NI
-1-1 -1-7 + 0-9 + 0-4 + 0-4 -1-8 + 0-4 -1-9 + 0-8 -1-6 -1-7 -0-4
-1-9 + 0-2
+ 1-0
-1-8
R
NI
BS Cs BS + Cs
Electrophoretic mnhilitv lllvUUlVT (cm/h)
oenetic locationf
Expression of enzyme*
Profile nf vl enzyme
* NI, not indudblc enzyme; I, inducible enzyme. t R, R factor-mediated; C, chromosomally mediated. \ CR, cephaloridine; other abbreviations of antibiotics are the same as those given in the footnote, to Table I.
E. coli E. coli E. coli Klebsiella Klebsiella Klebsiella S. marcescens S. marcescens E. aerogenes E. cloacae P. morganii P. morganii P. vulgaris P. vulgaris P. rettgeri P. stuartii P. stuartii
Organism
100 100 100 100 100 100 100 100 100 100 100 100 100
100
100 100 100
CRt
19 439 350 27 15 122 30 89 40 128 46 366 124 217 21 44 83
CF
44 0 0 9 37 0 116 190 76 52 0
157
44
65
41 0 44
CA
0 0 0 0 0 87 0 0 0
0
0 0 0 0 0 11 0
cu
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
CO
Relative rates of hydrolysis
Table IL Characteristics of P-lactamases from representative organisms of different resistance groups
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A. Vnye, J. Pijck and H. W. Van Landuyt
298 -20 20
B
15
10
C»
Ct
I?
BS+Ct
i= BS,
BS 3
Klabsiella
£ coll >20 20
BS2
B
mB
Ct
BS
£. clocc. S. marc
ms
'i =
15
10
i=
*= Ct
marc
BS
Ct Ct R m organ/I
BS
Ct
P vulgaris
BS BS Cs P reltg. Prom stuart//
• .cephalothio; O, cefamandole; Q, c«furoxlm«; D , cefoxltln Figure 1. Times from addition of antibiotic to recommencement of growth after lysis for selected organisms, representative for different enzymatic resistance classes. The profile of the main enzyme of each strain is likewise indicated in the Figure. I, incomplete lysis.
Discussion Gram-negative organisms, which are resistant against {Mactam antibiotics generally contain one or more of several different types of cell-bound p-lactamases (Jack & Richmond, 1970; Richmond & Sykes, 1973; Sykes & Matthew, 1976). Although resistance to these antibiotics appears to be also dominated by other resistance mechanisms, such as permeability barriers to the entry into the cells, enzymatic inactivation probably can be considered as the major defence mechanism of Gram-negative organisms
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BS
Activity of cefamandole, cefuroxime, cefoxitin
299
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against p-lactam antibiotics (Richmond & Curtis, 1974). In the present study we found production of different types of P-lactamases to be a valuable guide for the recognition and delineation of resistance classes among ampicillin resistant Enterobacteriaceae with respect to three conventional and three newer p-lactam antibiotics. Antibiotic susceptibility of these ampicillin resistant isolates was especially investigated with respect to 2 newer cephalosporins and to cefoxitin which were reported by several authors to display considerable broadening of their Gram-negative spectrum. Cefamandole was originally reported to be relatively resistant against TEM-type P-lactamases (Neu, 1974a). LateT investigations however indicated that its enzymatic stability was restricted to the typical cephalosporinases only (Richmond & Wotton, 1976). Our observations agreed with the latter concept: all broad spectrum enzymes encountered, whether chromosomally or R factor-mediated, inactivated the compound to a considerable degree, whereas cephalosporinases were mostly without effect, with the exception of those produced by Proteus vulgaris. In disc susceptibility tests, cefamandole proved to be active on many cephalothin resistant (mostly cephalosporinaseproducing) strains of E. coli, enterobacter, indole-positive proteus and providencia. However, the increased activity of cefamandole against these strains is generally associated with a high inoculum effect and a great difference between bactericidal and inhibitory levels (Neu, 1974a). Cefuroxime is a new P-lactamase stable cephalosporin, reported to have wide activity against Gram-negative bacilli, including enterobacter, some indole-positive proteus and Haemophilus influenzae (Eykyn, Jenkins, King & Phillips, 1976; O'Callaghan, Sykes, Ryan, Foord & Muggleton, 1976). In conventional susceptibility testing, we found the antibiotic to have a similar Gram-negative spectrum as cefamandole. However activity of cefuroxime is less influenced by inoculum effects, which can probably be accounted for by its higher stability against most enterobacterial P-lactamases. The spectrum of cefoxitin on the other hand included furthermore many strains of Serratia marcescens and Proteus vulgaris. The latter species was rather resistant against cefamandole and cefuroxime, in contrast to the other indole-positive proteus, probably as a result of the high inactivating potential of the cephalosporinases of this particular species against both compounds. In conventional susceptibility tests, cefoxitin did not demonstrate any significant activity against Enterobacter spp. However in the turbidimetric system, the antibiotic displayed a profound lytic effect and regrowth times that were only slightly shorter than those of cefamandole and cefuroxime. It is further worth noting that cefoxitin was active on many cephalothin resistant strains, which were simultaneously resistant to carbenicillin. The TEM-producing Serratia marcescens strains (56 % of the total number investigated) constitute one example. These strains, while invariably resistant to carbenicillin, were mostly susceptible to cefoxitin. Like many other authors (Neu, 19746; Onishi, Daoust, Zimmerman, Hendlin & Stapley, 1974; Richmond & Wotton, 1976; Darland & Birnbaum, 1977), we found cefoxitin to be completely resistant against most Gram-negative P-lactamases (Vuye & Pijck, 1978). From these observations it may be concluded that cefamandole, cefuroxime and cefoxitin, as compared with more conventional cephalosporins, show a considerable broadening of their Gram-negative activity profile. This property seems to be directly related with their increased stability against Gram-negative p-lactamases. Cefuroxime and cefoxitin appear to be stable against both cephalosporinases and broad spectrum enzymes, whereas cefamandole can resist the action of the former enzymes only. In this respect,
300
A. Voye, J. Pijck and H. W. Van Landuyt
determination of enzymatic resistance mechanisms can provide useful information for a rational choice of the most appropriate antibiotic. References
{Manuscript accepted 20 September 1978)
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Bauer, A. W., Kirby, W. M. M., Sherris, J. C. & Turck, M. Antibiotic susceptibility testing by a standardized single disk method. American Journal Clinical Pathology 45: 493-6 (1966). Darland, G. & Bimbaum, J. Cefoxitin resistance to beta-lactamase: a major factor for susceptibility of Bacteroides fragilis to the antibiotic. Antimicrobial Agents and Chemotherapy 11: 725-34 (1977). Eykyn, S., Jenkins, C , King, A. & Phillips, I. Antibacterial activity of cefuroxime, a new cephalosporin antibiotic, compared with that of cephaloridine, cephalothin, and cefamandole. Antimicrobial Agents and Chemotherapy 9: 690-5 (1976). Farrar, W. E. & Krause, J. M. Relationship between (5-lactamase activity and resistance of Enterobacter to cephalothin. Infection and Immunity 2: 610-6 (1970). Greenwood, D. Response profiles: a method of evaluating the activity of J)-lactam antibiotics against Enterobacteria. Chemotherapy 23: 11-8 (1977). Greenwood, D. & O'Grady, F. Comparison of the responses of Escherichia coli and Proteus mirabilis to seven fj-lactam antibiotics. Journal of Infectious Diseases 128: 211-22 (1973). Jack, G. W. & Richmond, M. H. A comparative study of eight distinctfr-lactamasessynthesized by Gram-negative bacteria. Journal of General Microbiology 61: 43-61 (1970). Neu, H. C. Cefamandole, a cephalosporin antibiotic with an unusually wide spectrum of activity. Antimicrobial Agents and Chemotherapy 6: 177-82 (1974a). Neu, H. C. Cefoxitin, a semisynthetic cephamycin antibiotic: antibacterial spectrum and resistance to hydrolysis by Gram-negative beta-lactamases. Antimicrobial Agents and Chemotherapy 6: 170-6 (19746). Novick, R. P. Microiodometric assay for penicillinase. Biochemical Journal 83: 236-40 (1962). O'Callaghan, C. H., Sykes, R. B., Ryan, D. M., Foord, R. D. & Muggleton, P. W. Cefuroxime —a new cephalosporin antibiotic. Journal of Antibiotics 29: 29-37 (1976). Onishi, H. R., Daoust, D. R., Zimmerman, S. B., Hendlin, D. & Stapley, E. O. Cefoxitin, a semisynthetic cephamycin antibiotic: resistance to beta-lactamase inactivation. Antimicrobial Agents and Chemotherapy 5: 38-48 (1974). Richmond, M. H. & Curtis, N. A. C. The interplay of P-lactamases and intrinsic factors in the resistance of Gram-negative bacteria to penicillins and cephalosporins. Annals of New York Academy of Sciences 235: 553-67 (1974). Richmond, M. H. & Sykes, R. B. The [Mactamases of Gram-negative bacteria and their possible physiological role. In Advances in Microbial Physiology. (Rose, A. H. & Tempest, D. W. Eds). Academic Press, London and New York (1973), Vol. 9, pp. 31-88. Richmond, M. H. & Wotton, S. Comparative study of seven cephalosporins: susceptibility to beta-lactamases and ability to penetrate the surface layers of Escherichia coli. Antimicrobial Agents and Chemotherapy 10: 219-22 (1976). Ross, G. W. & O'Callaghan, C. H. P-lactamase assays. In Methods in Enzymology. (Hash, J. H Ed.). Academic Press Inc., New York (1975), Vol. 43, pp 69-86. Sabath, L. D. & Finland, M. Resistance of penicillins and cephalosporins to P-lactamase from Gram-negative bacilli: some correlations with antibacterial activity. Annals of New York Academy of Sciences 145: 237-47 (1967). Smithies, O. Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults. Biochemical Journal 61: 629-41 (1955). Sykes, R. B. & Matthew, ML The (J-lactamases of Gram-negative bacteria and their r61e in resistance to 0-lactam antibiotics. Journal of Antimicrobial Chemotherapy 2:115-57 (1976) Vuye, A. & Pijck, J. Comparison of stabilities of cephalosporins against beta-lactamases from Gram-negative organisms. In Current Chemotherapy. (Siegenthaler, W. & Hithy, R., Eds). American Society for Microbiology, Washington D. C. (1978), Vol. 1, 492-4.
Comparative activity of two newer cephalosponns, cefoxitin, and cephalothin against selected Enterobacteriaceae and correlation with enzymatic resistance mechanisms
Department of Microbiology, Faculty of Pharmacy, University of Gent, Apotheekstraat 1, B-9000 Gent, Belgium
and H. W. Van Landnyt,
Department of Microbiology, General Hospital St-Jan, Brugge, Belgium
A comparative in vitro investigation of three newer P-lactam antibiotics (cefamandole, cefuroxime and cefoxitin) against ampicillin and/or cephalothin resistant Enterobacteriaceae was performed by disc diffusion testing and by continuous turbidimetric monitoring. Cefamandole displayed good activity against cephalosporinase-producing organisms, with the exception of serratia and Proteus vulgaris. Production of broad spectrum enzymes generally resulted in rapid inactivation of cefamandole. In contrast, cefuroxime and cefoxitin demonstrated high resistance against most enterobacterial P-lactamases. Cefoxitin was the only antibiotic with considerable activity against serratia. The same compound was inactive against enterobacter in conventional tests, but nevertheless did demonstrate a transient inhibitory effect in the turbidimetric system. Introduction
The cephalosporins have a wide spectrum of activity against both Gram-positive and Gram-negative organisms. However, conventional cephalosporins (cephalothin, cephaloridine, cephalexin, cefazolin) have only minor activity against certain Enterobacteriaceae such as serratia, enterobacter, indole-positive proteus and providencia. This resistance is primarily due to hydrolytic attack by different P-lactamases (Sabath & Finland, 1967; Farrar & Krause, 1970). Recently a number of new p-lactam antibiotics have been introduced which show a markedly wider spectrum of activity against Gram-negative bacilli. The most promising of these compounds are cefamandole and cefuroxime, which are cephalosporins and cefoxitin which belongs to the cephamycins. The purpose of this study was to compare the in vitro activity of these newer antibiotics with respect to a number of selected ampicillin and/or cephalothin resistant 0305-7453/79/030293+08 ?01.00/0
293 © 1979 The British Society for Antimicrobial Chemotherapy
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A. Vuye, J. Pijck,
294
A. Vuye, J. Pijck and H. W. Van Landuyt
Enterobacteriaceae. In order to provide a rationale for understanding some of the differences in antimicrobial activity of the compounds, enzymatic resistance mechanisms were characterized in all tested organisms. Materials and methods Bacterial strains
Investigation of enzymatic resistance mechanisms Preparation of enzymes. Crude extracts were obtained by sonic disruption of cell suspensions, followed by removal of cell debris by centrifugation. Characterization of $-lactamases. Electrophoretic separations of the crude enzymes were performed at pH 8-5 by the starch-gel method of Smithies (1955). P-Lactamases were recorded as broad spectrum (BS) enzymes or cephalosporinases (Cs), by comparing the relative rates of hydrolysis of benzylpenicillin and cephaloridine at a substrate concentration of 5 mM. A modification of the micro-iodometric method ofNovick (1962) was used for determination of enzymatic activity with these compounds as substrate. Relative rates of enzymatic hydrolysis of the cephalosporins at a substrate concentration of 10~4 M were determined by the spectrophotometric method, described by Ross & O'Callaghan (1975). Transfer of R-plasmids. Organisms were mated with a rifampicin or nalidixic acid resistant substrain of E. coli K12. Transconjugants were selected on antibiotic containing selective media, purified, and tested for drug resistance and production of (i-lactamases. Resistance to ampicillin in the transconjugants obtained in this way, was primarily due to fl-lactamase production, and provided evidence of the extrachromosomal genetic basis for p-lactamase formation. When failure of conjugational transfer of the pMactamase was observed, the enzyme was scored as probably chromosomally mediated. However, to locate unequivocally a gene on the bacterial chromosome, more sophisticated methods are needed. Susceptibility tests Antibiotic susceptibility of all organisms tested was determined on Mueller-Hinton agar by the standard disc diffusion method of Bauer, Kirby, Sherris & Turck (1966). Organisms which gave a zone of inhibition of 18 mm diameter or greater, obtained with 0-6 cm discs containing 30 ug of the respective cephalosporins, were designated susceptible to these compounds. For ampicillin (10 ug) and carbenicillin (100 ug), 14 and 23 mm were respectively taken as the minimum inhibition zone diameters, representing a sensitive response.
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Most of the strains were recently isolated from patients in St-Jan's Hospital, Brugge. The selection of Escherichia coli and klebsiella strains took place on the basis of preliminary susceptibility tests with ampicillin and cephalothin. Only strains resistant to one or both of these compounds were further investigated. In general, the remaining species investigated (Enterobacter aerogenes, E. cloacae, Serratia marcescens, Proteus morganii, P. vulgaris, P. rettgeri and Providencia stuartii) were resistant to cephalothin. Consequently, all strains of these species were selected for further investigation.
Activity of cefamandole, cefaroxime, cefoxitin
295
Antibiotics Cephalothin and cefamandole were provided by Eli Lilly and Co., cefuroxime by Glaxo and cefoxitin by Merck, Sharp and Dohme. Results The profiles of the enzymes produced in different Gram-negative species are indicated in Table I, together with disc diffusion susceptibilities to 2 penicillins, 3 cephalosporins and cefoxitin. Characteristics of the enzymes are scored in Table n . Production of broad spectrum enzymes, which were either chromosomally or R factor-mediated was observed in all genera, except enterobacter. Among the klebsiella strains, one isolate was found that produced a rather exceptional enzyme (BS3) which could not be identified with one of the enzyme types, described by Richmond & Sykes (1973). This enzyme was a constitutive broad spectrum P-lactamase, with an electrophoretic mobility slightly lower than that of EUa (TEM) enzyme. It hydrolysed to a considerable degree all fJ-lactam antibiotics, with the exception of cefoxitin. Typical cephalosporinases, which could never be transferred by conjugation, were found in all genera except klebsiella. The Cs enzymes produced by E. coli differed from other cephalosporinases in that they were invariably constitutive. All strains of Proteus rettgeri were found to produce broad spectrum enzymes in addition to their speciesspecific cephalosporinases. Broad spectrum enzymes, whether chromosomally or R factor-mediated invariably caused resistance to both ampicillin and carbenicillin. Production of a cephalosporinase was generally indicative for high-degree resistance against cephalothin, regardless of the genus concerned. The newer compounds (cefamandole, cefuroxime and cefoxitin) were active on a great number of the ampicillin and/or cephalothin resistant isolates. In general, cefoxitin had the most reliable activity but it was ineffective against enterobacter, when susceptibility was determined with conventional methods. Cefoxitin furthermore proved to be the only fl-lactam antibiotic with reliable activity against Serratia marcescens. Rates of hydrolysis of the different cephalosporins [relative to hydrolysis of cephaloridine ( = 100)] are illustrated in Table EL Cefoxitin was clearly the most stable compound
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From each of the different enzymatic resistance groups observed, a limited number of strains were selected for further susceptibility testing by continuous turbidimetric monitoring. The turbidimetric system consisted of six stirred cultures in Mueller-Hinton broth, which were continuously monitored in a Bonet-Maury biophotometer (Jobin Yvon instruments). Antibiotics were added at the same point in the logarithmic phase, when the optical density was equivalent to a viable count of about 1 x 107 bacteria/ml. These conditions differ markedly from those of conventional susceptibility tests, but it may be argued that the denser population represents the numbers of bacteria in vivo more closely (Greenwood & O'Grady, 1973). The concentration of antibiotic was fixed at the cut-off point of 32 ug/ml, representing the maximum blood level concentration of most cephalosporins. The time elapsing between addition of antibiotic and lysis of the culture and the time before regrowth occurred were both noted. In this way information can be obtained on both the intrinsic activity of the compounds and their susceptibility to enzymatic inactivation (Greenwood, 1977).
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296
Table L Distribution of p-lactamases among ampicillin and/or cephalothin resistant Enterobacteriaceae with indication of susceptibility to different 3-lactam antibiotics
Organ isn:i (total no. of strains) E. coli
(39)
P-lactamases produced BS*
Cst
Klebsiella
(22)
P. morganii
(13)
P. vulgaris
(10)
P. rettgeri Prov. stuartii
(7) (9)
No. of strains resistant to:
enzyme(s) 29 7 3 10 11 1 3 11 10 8 2 11 1 9 7 6 3
APt CB CF CA CU CO 29 29 19 7 0 7 3 3 3 10 10 9 11 11 3 1 1 1 0 0 3 1 11 3 10 9 10 7 0 8 2 2 2 6 0 11 1 1 1 9 1 9 7 7 6 6 6 4 2 0 3
2 0 4 1 1 1 2 2 3 0 2 0 1 0 1 1 0 1 1 2 0 1 1 10 1 10 10 8 7 1 1 2 0 1 2 0 1 0 1 8 6 1 1 0 3 0 0 0 0 0 0
* BS, broad spectrum enzyme t Cs, cephalosporinase. \ AP, ampicillin. CB, carbenicillin; CF, cephalothin; CA, cefamandole; CU, cefuroxinie; CO, cefoxitin.
since none of the investigated enzymes was able to hydrolyze this antibiotic. Cefamandole was found to be rapidly hydrolyzed by broad spectrum enzymes, but stable against cephalosporinases, with the exception of those from Proteus vulgaris. Figure 1 illustrates graphically the time between the addition of antibiotic and the recommencement of growth after lysis as shown by turbidimetric measurements. Trie strains selected for the figure were representative for the average response of isolates belonging to the same enzymatic resistance class. Cefamandole, like cephalothin, showed only a transient inhibitory effect against most organisms with R factor-mediated enzymes. With several strains (e.g. most strains of serratia) cefamandole and cefuroxime displayed only incomplete lytic effect, indicating lack of intrinsic activity (turbidity fell to an intermediate level, which was maintained during variable time periods). Biophotometer tracings obtained with different enterobacter strains showed that cefamandole and cefuroxime suppressed growth only slightly longer than cefoxitin. The extent of lysis before regrowth occurred was similar for the three compounds. Thus, lack of susceptibility of enterobacter to cefoxitin demonstrated in conventional susceptibility tests could not be corroborated by turbidimetric monitoring under the defined conditions. The mechanism of cefoxitin resistance in enterobacter clearly needs further investigation. Intrinsic activity as shown by rapidity and extent of lysis was generally highest for cefoxitin. Cefuroxime as a rule was less intrinsically active than cefamandole.
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E. aerogenes (3) E. cloacae (11) S. marcescens (18)
BS + Cs BS! BS, BS. Cs Cs BS + Cs Cs BS + Cs Cs BS + Cs Cs BS + Cs BS Cs
No. of strains with inHipfltcH
9/53 23/77 29/85 49 65 9/78 10 23 34 23/53 23/80 33 9/57 64 19B 23/41 101
R C C R C C C
NI NI
NI NI I I I NI I NI I NI NI I
BS, BS.
BS, BS
Cs
BS BS
Cs
BS
Cs
Cs BS
Cs Cs
C
R R
C
R
C
R
C
NI
-1-1 -1-7 + 0-9 + 0-4 + 0-4 -1-8 + 0-4 -1-9 + 0-8 -1-6 -1-7 -0-4
-1-9 + 0-2
+ 1-0
-1-8
R
NI
BS Cs BS + Cs
Electrophoretic mnhilitv lllvUUlVT (cm/h)
oenetic locationf
Expression of enzyme*
Profile nf vl enzyme
* NI, not indudblc enzyme; I, inducible enzyme. t R, R factor-mediated; C, chromosomally mediated. \ CR, cephaloridine; other abbreviations of antibiotics are the same as those given in the footnote, to Table I.
E. coli E. coli E. coli Klebsiella Klebsiella Klebsiella S. marcescens S. marcescens E. aerogenes E. cloacae P. morganii P. morganii P. vulgaris P. vulgaris P. rettgeri P. stuartii P. stuartii
Organism
100 100 100 100 100 100 100 100 100 100 100 100 100
100
100 100 100
CRt
19 439 350 27 15 122 30 89 40 128 46 366 124 217 21 44 83
CF
44 0 0 9 37 0 116 190 76 52 0
157
44
65
41 0 44
CA
0 0 0 0 0 87 0 0 0
0
0 0 0 0 0 11 0
cu
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
CO
Relative rates of hydrolysis
Table IL Characteristics of P-lactamases from representative organisms of different resistance groups
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A. Vnye, J. Pijck and H. W. Van Landuyt
298 -20 20
B
15
10
C»
Ct
I?
BS+Ct
i= BS,
BS 3
Klabsiella
£ coll >20 20
BS2
B
mB
Ct
BS
£. clocc. S. marc
ms
'i =
15
10
i=
*= Ct
marc
BS
Ct Ct R m organ/I
BS
Ct
P vulgaris
BS BS Cs P reltg. Prom stuart//
• .cephalothio; O, cefamandole; Q, c«furoxlm«; D , cefoxltln Figure 1. Times from addition of antibiotic to recommencement of growth after lysis for selected organisms, representative for different enzymatic resistance classes. The profile of the main enzyme of each strain is likewise indicated in the Figure. I, incomplete lysis.
Discussion Gram-negative organisms, which are resistant against {Mactam antibiotics generally contain one or more of several different types of cell-bound p-lactamases (Jack & Richmond, 1970; Richmond & Sykes, 1973; Sykes & Matthew, 1976). Although resistance to these antibiotics appears to be also dominated by other resistance mechanisms, such as permeability barriers to the entry into the cells, enzymatic inactivation probably can be considered as the major defence mechanism of Gram-negative organisms
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BS
Activity of cefamandole, cefuroxime, cefoxitin
299
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against p-lactam antibiotics (Richmond & Curtis, 1974). In the present study we found production of different types of P-lactamases to be a valuable guide for the recognition and delineation of resistance classes among ampicillin resistant Enterobacteriaceae with respect to three conventional and three newer p-lactam antibiotics. Antibiotic susceptibility of these ampicillin resistant isolates was especially investigated with respect to 2 newer cephalosporins and to cefoxitin which were reported by several authors to display considerable broadening of their Gram-negative spectrum. Cefamandole was originally reported to be relatively resistant against TEM-type P-lactamases (Neu, 1974a). LateT investigations however indicated that its enzymatic stability was restricted to the typical cephalosporinases only (Richmond & Wotton, 1976). Our observations agreed with the latter concept: all broad spectrum enzymes encountered, whether chromosomally or R factor-mediated, inactivated the compound to a considerable degree, whereas cephalosporinases were mostly without effect, with the exception of those produced by Proteus vulgaris. In disc susceptibility tests, cefamandole proved to be active on many cephalothin resistant (mostly cephalosporinaseproducing) strains of E. coli, enterobacter, indole-positive proteus and providencia. However, the increased activity of cefamandole against these strains is generally associated with a high inoculum effect and a great difference between bactericidal and inhibitory levels (Neu, 1974a). Cefuroxime is a new P-lactamase stable cephalosporin, reported to have wide activity against Gram-negative bacilli, including enterobacter, some indole-positive proteus and Haemophilus influenzae (Eykyn, Jenkins, King & Phillips, 1976; O'Callaghan, Sykes, Ryan, Foord & Muggleton, 1976). In conventional susceptibility testing, we found the antibiotic to have a similar Gram-negative spectrum as cefamandole. However activity of cefuroxime is less influenced by inoculum effects, which can probably be accounted for by its higher stability against most enterobacterial P-lactamases. The spectrum of cefoxitin on the other hand included furthermore many strains of Serratia marcescens and Proteus vulgaris. The latter species was rather resistant against cefamandole and cefuroxime, in contrast to the other indole-positive proteus, probably as a result of the high inactivating potential of the cephalosporinases of this particular species against both compounds. In conventional susceptibility tests, cefoxitin did not demonstrate any significant activity against Enterobacter spp. However in the turbidimetric system, the antibiotic displayed a profound lytic effect and regrowth times that were only slightly shorter than those of cefamandole and cefuroxime. It is further worth noting that cefoxitin was active on many cephalothin resistant strains, which were simultaneously resistant to carbenicillin. The TEM-producing Serratia marcescens strains (56 % of the total number investigated) constitute one example. These strains, while invariably resistant to carbenicillin, were mostly susceptible to cefoxitin. Like many other authors (Neu, 19746; Onishi, Daoust, Zimmerman, Hendlin & Stapley, 1974; Richmond & Wotton, 1976; Darland & Birnbaum, 1977), we found cefoxitin to be completely resistant against most Gram-negative P-lactamases (Vuye & Pijck, 1978). From these observations it may be concluded that cefamandole, cefuroxime and cefoxitin, as compared with more conventional cephalosporins, show a considerable broadening of their Gram-negative activity profile. This property seems to be directly related with their increased stability against Gram-negative p-lactamases. Cefuroxime and cefoxitin appear to be stable against both cephalosporinases and broad spectrum enzymes, whereas cefamandole can resist the action of the former enzymes only. In this respect,
300
A. Voye, J. Pijck and H. W. Van Landuyt
determination of enzymatic resistance mechanisms can provide useful information for a rational choice of the most appropriate antibiotic. References
{Manuscript accepted 20 September 1978)
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Bauer, A. W., Kirby, W. M. M., Sherris, J. C. & Turck, M. Antibiotic susceptibility testing by a standardized single disk method. American Journal Clinical Pathology 45: 493-6 (1966). Darland, G. & Bimbaum, J. Cefoxitin resistance to beta-lactamase: a major factor for susceptibility of Bacteroides fragilis to the antibiotic. Antimicrobial Agents and Chemotherapy 11: 725-34 (1977). Eykyn, S., Jenkins, C , King, A. & Phillips, I. Antibacterial activity of cefuroxime, a new cephalosporin antibiotic, compared with that of cephaloridine, cephalothin, and cefamandole. Antimicrobial Agents and Chemotherapy 9: 690-5 (1976). Farrar, W. E. & Krause, J. M. Relationship between (5-lactamase activity and resistance of Enterobacter to cephalothin. Infection and Immunity 2: 610-6 (1970). Greenwood, D. Response profiles: a method of evaluating the activity of J)-lactam antibiotics against Enterobacteria. Chemotherapy 23: 11-8 (1977). Greenwood, D. & O'Grady, F. Comparison of the responses of Escherichia coli and Proteus mirabilis to seven fj-lactam antibiotics. Journal of Infectious Diseases 128: 211-22 (1973). Jack, G. W. & Richmond, M. H. A comparative study of eight distinctfr-lactamasessynthesized by Gram-negative bacteria. Journal of General Microbiology 61: 43-61 (1970). Neu, H. C. Cefamandole, a cephalosporin antibiotic with an unusually wide spectrum of activity. Antimicrobial Agents and Chemotherapy 6: 177-82 (1974a). Neu, H. C. Cefoxitin, a semisynthetic cephamycin antibiotic: antibacterial spectrum and resistance to hydrolysis by Gram-negative beta-lactamases. Antimicrobial Agents and Chemotherapy 6: 170-6 (19746). Novick, R. P. Microiodometric assay for penicillinase. Biochemical Journal 83: 236-40 (1962). O'Callaghan, C. H., Sykes, R. B., Ryan, D. M., Foord, R. D. & Muggleton, P. W. Cefuroxime —a new cephalosporin antibiotic. Journal of Antibiotics 29: 29-37 (1976). Onishi, H. R., Daoust, D. R., Zimmerman, S. B., Hendlin, D. & Stapley, E. O. Cefoxitin, a semisynthetic cephamycin antibiotic: resistance to beta-lactamase inactivation. Antimicrobial Agents and Chemotherapy 5: 38-48 (1974). Richmond, M. H. & Curtis, N. A. C. The interplay of P-lactamases and intrinsic factors in the resistance of Gram-negative bacteria to penicillins and cephalosporins. Annals of New York Academy of Sciences 235: 553-67 (1974). Richmond, M. H. & Sykes, R. B. The [Mactamases of Gram-negative bacteria and their possible physiological role. In Advances in Microbial Physiology. (Rose, A. H. & Tempest, D. W. Eds). Academic Press, London and New York (1973), Vol. 9, pp. 31-88. Richmond, M. H. & Wotton, S. Comparative study of seven cephalosporins: susceptibility to beta-lactamases and ability to penetrate the surface layers of Escherichia coli. Antimicrobial Agents and Chemotherapy 10: 219-22 (1976). Ross, G. W. & O'Callaghan, C. H. P-lactamase assays. In Methods in Enzymology. (Hash, J. H Ed.). Academic Press Inc., New York (1975), Vol. 43, pp 69-86. Sabath, L. D. & Finland, M. Resistance of penicillins and cephalosporins to P-lactamase from Gram-negative bacilli: some correlations with antibacterial activity. Annals of New York Academy of Sciences 145: 237-47 (1967). Smithies, O. Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults. Biochemical Journal 61: 629-41 (1955). Sykes, R. B. & Matthew, ML The (J-lactamases of Gram-negative bacteria and their r61e in resistance to 0-lactam antibiotics. Journal of Antimicrobial Chemotherapy 2:115-57 (1976) Vuye, A. & Pijck, J. Comparison of stabilities of cephalosporins against beta-lactamases from Gram-negative organisms. In Current Chemotherapy. (Siegenthaler, W. & Hithy, R., Eds). American Society for Microbiology, Washington D. C. (1978), Vol. 1, 492-4.
