- Email: [email protected]
OBSERVATIONS ON THE NATURE, DISTRIBUTION, AND SIGNIFICANCE OF CEPHALOSPORINASE
1399
(1952). Tumour antigens have been serum of patients with various and have been shown to be capable diseases neoplastic the formation of antibodies (Makari 1958). of provoking skin sensitivity in a Dao demonstrated and Grace (1959)
myositis by
Curtis
Preliminary
demonstrated in the
with carcinoma of the breast and dermatomyositis to an aqueous extract of her own tumour, and Curtis et al. (1961) in a similar case showed that the antibodies were of a circulatory type. The association of Hashimoto’s disease in our case is perhaps of interest; for of the many diseases thought to be autoimmune in origin, Hashimoto’s thyroiditis is the one woman
whose credentials
beyond reproach. Although we cannot trace a previous report linking Hashimoto’s disease with carcinoma and dermatomyositis or a neuromyopathy, Brain and Henson (1958) note that 3 of the 15 women
are
in their series of
cases
of carcinomatous
myopathy had myxoedema. These patients
were
neuro-
mostly
under observation before there were autoimmune tests for thyroid antibodies and before it was realised that Hashimoto’s disease often ended in myxoedema. The thyroid of 1 of these patients is described in a necropsy report (case 1) by Henson et al. (1954), and the focal lymphocytic infiltration, degenerating acini, and extensive fibrous replacement are consistent with autoimmune thyroiditis. It is clear, however, that if thyroiditis had been the cause of the myxcedema it must have developed long before the carcinomatous neuropathy, since the patient had been taking thyroid for several years. Case 2 in the same series was a woman with a bronchial carcinoma, and although she was not myxcedematous, the thyroid gland at necropsy showed degeneration of acini and focal lymphatic infiltration. In another series, of patients with carcinomatous neuropathy reported by Brain et al. (1951), there was 1 woman (case 1) who was not myxoedematous at the time of her death, but her thyroid gland showed lymphocytic infiltration to a remarkable degree " though less pronounced than in Riedel’s struma. These findings suggest Hashimoto’s disease, and it seems not unlikely that this may have been the cause of the myxoedema in the other two women reported by Brain and Henson (1958). Hall et al. (1962) suggested that the tendency to form thyroid antibodies may be an inherited characteristic; and if the association between Hashimoto’s disease and carcinomatous neuropathy is confirmed and is found to be greater than could be explained by chance, it would lend support to the speculation that carcinomatous neuromyopathy is related to a constitutional reaction of some patients to a neoplasm rather than to any toxic or metabolic effects of the tumour. Nevertheless, Brain and Henson (1958) rightly warn against any facile interpretation of carcinomatous neuromyopathy, and the difference in sex incidence between this condition and Hashimoto’s disease makes the suggested association of doubtful significance, and offers no explanation for the higher incidence of neuromyopathy in certain tumours. "
histological
Communications
OBSERVATIONS ON THE NATURE, DISTRIBUTION, AND SIGNIFICANCE OF
CEPHALOSPORINASE cephalosporins are a series of antibiotics with a common nucleus containing a fused P-lactam dihydrothiazine ring very similar to the fused p-lactam thiazolidine ring of the nucleus of the penicillins. The general properties of these two groups of antibiotics are very much the same, and their mode of action is probably identical. The cephalosporins have recently been completely reviewed by Abraham.1 The promising features which may be exploited for clinical purposes include a suggested absence of cross-allergenicity with the penicillins,2 a broader gram-negative spectrum,3 and resistance to staphylococcal penicillinase.4 Penicillin is almost unique among antibiotics in clinical use in that resistance of bacteria to it is very often, and in some species practically always, mediated by enzymic destruction of the drug. Of chief clinical interest has been the P-lactamase, because of its close association with the resistance of staphylococci. Penicillin acylases, which split off the side-chain but do not affect the nucleus, also exist and contribute to resistance, particularly in the gram-negative species.5 We report here the production (by a species of aerobacter) of an enzyme, apparently a P-lactamase, which is highly active against cephalosporin C (ex-amino adipyl cephalosporin) but which has little if any such action against penicillin G. We have also observed that enzymes degrading the cephalosporins, whether j3-lactamase(s) or otherwise, are widely distributed within the enterobacteriacex, and that in some species their presence is remarkably constant. In general, the most resistant species are among those which produce these enzymes, whereas in the sensitive species we have not been able to THE
detect their presence. EXPERIMENTAL STUDIES
A strain of Enterobacter cloacae (Aerobacter subgroup A) isolated from a blood-culture was found to be tolerant to approximately 10,000 flg. per ml. of cephalosporin C. Initial investigation revealed that it produced a filterable enzyme which rapidly inactivated the antibiotic. 61 An enzyme concentrate (nitrogen content, 13-2%) was prepared from the cell-free culture supernatant by precipitation with saturated ammonium sulphate. This preparation was tested against the following substrates: cephalosporin C, deacetyl cephalosporin C, cephalosporin G (benzyl cephaloAbraham, E. P. Pharmacol. Rev. 1962, 14, 473. Stewart, G. T. Lancet, 1962, i, 509. Chauvette, R. R., Flynn, E. H., Jackson, B. G., Lavagnino, E. R., Morin, R. B., Mueller, R. A., Pioch, R. P., Roeske, R. W., Ryan, C. W., Spencer, J. I., Van Heyningen, E. J. Amer. chem. Soc. 1962, 84, 3401. 4. Jago, M., Heatley, N. G. Brit. J. Pharmacol. 1961, 16, 170. 5. English, A. R., McBride, T. J., Huang, H. T. Proc. Soc. exp. Biol., N.Y. 1960, 104, 547. 6. Fleming, P. C. Canad. J. publ. Hlth, 1963, 54, 47 (abstract). 7. Fleming, P. C., Goldner, M. Fed. Proc. 1963, 22, 206 (abstract no. 274).
1. 2. 3.
Summary A
of 52 had Hashimoto’s disease, dermatomyositis, and carcinoma. Dermatomyositis and carcinomatous neuromyopathy resemble each other in many ways. An association between Hashimoto’s disease and carcinomatous neuromyopathy is suggested and discussed in relation to theories of the nature of the neuromyopathy. woman
W. R.
(1963) Lancet, i, 179. Daniel, P. M., Greenfield, J. G. (1951) J. Neurol. Psychiat. 14, 59. Henson, R. A. (1958) Lancet, ii, 971. Croft, P. B., Wilkinson, M. (1963) ibid. i, 184. Curtis, A. C., Blaylock, H. C., Harrell, E. R. (1952) J. Amer. med. Ass. 150, 844. Heckaman, J. H., Wheeler, A. H. (1961) ibid. 178, 571. Grace, J. T., Dao, T. L. (1959) Cancer, 12, 648. Hall, R., Saxena, K. M., Owen, S. A. (1962) Lancet, ii, 1291. Henson, R. A., Russell, D. S., Wilkinson, M. (1954) Brain, 77, 82. Makari, J. G. (1958) Brit. med. J. ii, 359.
Brain, —
—
—
1400 and penicillin G. A method modified from those of Perret and Tucker 9 was used to ascertain the number of iodine equivalents per mole consumed after the complete alkaline and enzymic hydrolysis of the antibiotics (see table), and to assay the activity of the enzyme concentrate. Infra-redabsorption studies on the lyophilised incubation mixtures were performed both by the KBr-pellet and by the Nujol-mull methods to observe changes in the &bgr;-lactam structure of the
sporin),
8
’
antibiotics. The criteria for identification of enterobacteriaceae were as described by Edwards and Ewing.1O In the penicillins, hydrolysis of the p-lactam ring, either by dilute alkali or by enzyme (p-lactamase) activity, is known to be accompanied by an uptake of 7-9 equivalents of iodine The complete hydrolysis of penicillin G by the aerobacter-enzyme concentrate is also associated with a similar iodine uptake (see table), and this suggests that the penicillinase activity detected by this means is of the lactamase type. The P-lactam ring of cephalosporin C is probably also opened by dilute alkaline hydrolysis,12 and an iodine uptake of 4 equivalents has been reported for this reaction." We confirm this finding and note similar values for deacetyl cephalosporin C and for cephalosporin G. As the results of the enzymic and alkaline hydrolysis of the cephalosporins show such close agreement, it seems likely that the cephalosporinase activity of the concentrate, detected by this means, is also of the p-lactam type. Deacetylation of the acetoxyl side-chain, however, which IODINE
EQUIVALENTS
"
DISTRIBUTION OF
CEPHALOSPORINASES
"
16 was
The membrane method of Knox and Smith modified study the distribution of cephalosporinases and penicillinases in over a thousand strains of enterobacteriacese and associated organisms. This technique involves whole living organisms and detects cell-bound as well as filterable enzyme activity. As used here, it determines only the ability of the organism to produce acid degradation products from the substrate used. It is important to appreciate, therefore, that the terms cephalosporinase and penicillinase used below refer not only to cephalosporin and penicillin &bgr;-Iactamase activity but also to cephalosporin and penicillin acylase and to cephalosporinesterase (deacetylating) activity should these be present. Any of these enzymes may occur singly or in combination.
to
Technique Single colonies of the organism under test were grown overnight, suitably spaced on the surface of a ’Millipore’ membrane laid on a nutrient-agar plate. The membrane was then removed and placed on a Whatman no. 1 filterpaper saturated with a neutralised solution containing cephalosporin C (25,000 ij!.g. per ml.) or penicillin G (25,000 units per ml.), and 2% Andrade’s indicator. Membrane and filterpaper were then incubated at a suitable temperature and examined from time to time for the appearance of a red colour in or about the colony. A control omitting the substrate and using Andrade’s indicator alone was always included. All membranes were observed for six hours. Strong positive reactions showed up within twenty minutes. The acid reaction produced from cephalosporin C, unlike that from penicillin G, is apparently not stable as the colour usually fades within an hour of its appearance. RESULTS
The
organisms placed in one of three groups to their according ability to form acid from cephalosporin C by the membrane technique:
*
Except for deacetyl cephalosporin C, alkaline hydrolysis consumed more iodine than did enzymic hydrolysis.
a
little
is found in the cephalosporins but not in the penicillins, is not excluded. If present here, it does not account for any significant iodine absorption since the iodine equivalents are the same after enzymic hydrolysis of cephalosporin C and deacetyl cephalosporin C (see table).
Infra-red Absorption Studies Direct evidence that the p-lactam ring is opened during enzymic hydrolysis of penicillin G and cephalosporin C was provided by observing the disappearance of the infra-red at 5-62 !L after incubation with the enzyme This band is considered to be characteristic of a part of the intact fused &bgr;-Iactam ring.14 Specific Activities The values of the specific P-lactamase activity of the enzyme concentrate for the different substrates, measured iodometrically, and expressed as microgrammes of antibiotic hydrolysed per minute per milligramme dry weight of concentrate, are:
absorption band concentrate.
were
Group 1.-geneva and species, strains of which were consistently positive: Aerobacter (subgroups A, B, and C), serratia, hafnia, Proteus morganii, and Pseudomonas aeruginosa. Group 11.-Genera and species, strains of which were consistently negative: Salmonella, klebsiella (all species), Pr. mirabilis.
Group I11.-Genera and species, some strains of which were positive and others negative: Pr. vulgaris, Pr. rettgeri, Escherichia coli, Shigella sonnei, and some other Shigella species. Relationship of Cephalosporinase Production to Resistance Group I.-These strains were usually tolerant to several hundred or thousands of microgrammes per millilitre. Group 77.—These were usually sensitive to 5-50 g. per ml. Group 777.—In some species such as Sh. sonnei, cephalosporinase production was obviously related to resistance. In other species, such as Pr. vulgaris which is usually highly resistant, this resistance could not be correlated with ability to produce cephalosporinase. Relationship of Penicillinase to Cephalosporinase Production As determined by the membrane technique, there was no clear association between cephalosporinase and penicillinase activity. Organisms were isolated which showed penicillinase or cephalosporinase activity only, both, or neither. DISCUSSION
The activity of the enzyme concentrate against cephalosporin C very greatly exceeds its activity against penicillin G. We have not yet determined whether both these activities are due to the same or separate enzymes. Perret, C. J. Nature, Lond. 1954, 174, 1012. Tucker, R. G. ibid. 1934, 173, 85. 10. Edwards, P. R., Ewing, W. H. Identification of Enterobacteriaceæ. 8. 9.
Minneapolis, 1962. 11. Alicino, J. F. Industr. Engng Chem. (Anal.) 1946, 18, 619. 12. Crompton, B., Jago, M., Crawford, K., Newton, G. G. F., Abraham, 13. 14.
E. P. Biochem. J. 1962, 83, 52. Alicino, J. F. Anal. Chem. 1961, 33, 648. Abraham, E. P., Newton, G. G. F. Biochem. J. 1956, 62, 651.
Abraham and Newton,14 and later Crompton et al.,12 reported that culture filtrates of Bacillus cereus showed cephalosporinase (p-lactamase) in addition to penicillinase ((3-lactamase) activity. They noted that the activity of these filtrates was low against cephalosporin C, deacetyl cephalosporin C, and cephalosporin G, compared to penicillin G. Moreover, purified penicillinase prepared from these filtrates showed even less relative cephalosporinase activity. In contradistinction, both our aerobacter filtrate and the enzyme preparation derived from it were highly active against cephalosporin C, but they 15.
Knox, R., Smith, J. T. Nature, Lond. 1961, 191, 926.
1401 or little activity, respectively, against penicillin small amount of penicillinase activity present in The G. the concentrate could be derived from autolysed bacterial cells since it was shown by the membrane technique that this strain gave both strong penicillinase and cephalosporinase reactions. The varying activity of the enzyme against different cephalosporins suggests the possible development of
showed no
" cephalosporinase-resistant " cephalosporins. With the use of cephalosporins clinically,6 16 knowledge of the existence and distribution of inactivating enzymes is of obvious importance. The regular presence or absence of cephalosporinase in certain species may also be of "
"
taxonomic value. The
The Research Institute, Hospital for Sick Children, Toronto, Canada
P. C. FLEMING M.B.
Lond.
M. GOLDNER PH.D.
Connaught Medical Research Laboratories, University of Toronto
Montreal
D. G. GLASS B.SC.
McMaster
SPLENIC-TO-FEMORAL AND AXILLARY-TO-FEMORAL BYPASS GRAFTS IN DIFFUSE ATHEROSCLEROTIC OCCLUSIVE DISEASE ALTHOUGH restoration of arterial flow in atherosclerotic occlusive disease has now become standardised and
relatively simple, the salvage of legs in elderly or poor-risk patients with impending or overt gangrene still poses a difficult problem. In about half the cases, combined aorto-iliac and femoro-popliteal occlusions are found, and there is significant disease of the popliteal artery and its branches in almost 40%.1’ 18 In such cases satisfactory revascularisation of the legs entails a major procedurenamely, aorta-to-femoral-to-popliteal bypass graft 19 or aorto-iliac endarterectomy combined with femoral-toSociety for Microbiology. Antimicrobial Agents and Chemotherapy. Ann Arbor, 1962 (in the press). Louw, J. H., Roberts, W. M. S. Afr. med. J. 1961, 35, 346, 367, 385. Morris, G. C., Jr., Wheeler, C. G., Crawford, E. S., Cooley, D. A., De Bakey, M. E. Surgery, 1962, 51, 50. Morris, G. C., Jr., Edwards, W., Cooley, D. A., Crawford, E. S., De Bakey, M. E. Arch. Surg. 1961, 82, 32.
16. American 17. 18. 19.
These operations may, however, be unwise because of the poor condition of the patient, or impossible because of the extent of the disease. The surgeon, faced with the risk of a probable " amputation for mortification ", may be obliged to perform a compromise operation which will provide satisfactory, although not ideal, revascularisation. This can be achieved by limiting or modifying either the distal or the proximal extent of.the standard reconstructive procedure and combining it with sympathectomy when possible." 8-23
popliteal bypass graftsY
DISTAL LIMITATION
The value of simply restoring an adequate blood-flow to the profunda femoris artery in patients with combined aortò-iliac and femoro-popliteal occlusions has been well established. We have achieved this mainly by aorto-iliac endarterectomy with 70% success 17; others have claimed even better results with aorta-to-femoral bypass grafts combined with sympathectomy.120 The profunda femoris artery almost always remains remarkably free of atherosclerosis, and even if the common femoral artery appears completely occluded on arteriography, the patient should not be denied the benefit of this type of operation, with or without concomitant sympathectomy. 1-9 20 PROXIMAL LIMITATION
patients a good pulsatile flow is present in the aorta above the occlusion; hence anastomosis of a graft to the aorta or aorto-iliac endarterectomy is usually possible. But sometimes, because of extensive calcific or ulcerating disease, " irremovable aneurysms ", or fibrosis due to failure of previous vascular operations, these procedures are impossible or unduly hazardous. Moreover, operations on the aorta and iliac arteries have a high mortality-rate and morbidity-rate.24 A more suitable or accessible artery for the feeding vessel " may have to be used. The splenic artery has been used by several workers,25 and we have used it in 4 patients with remarkable success In
most
"
20. 21. 22. 23. 24. 25.
Leeds, F. H., Gilfillan, R. S. ibid. p. 25. McCaughan, J. J., Jr., Kahn, S. F. Ann. Surg. 1960, 151, Vetto, R. M. Surgery, 1962, 52, 342. Warren, R. Arch. Surg. 1956, 72, 57. Cockett, F. B., Maurice, B. A. Brit. med. J. 1962, i, 353. Freeman, N. E., Leeds, F. H. Surgery, 1953, 34, 1021.
BYPASS OPERATIONS IN VASCULAR INSUFFICIENCY
26.
(1952). Tumour antigens have been serum of patients with various and have been shown to be capable diseases neoplastic the formation of antibodies (Makari 1958). of provoking skin sensitivity in a Dao demonstrated and Grace (1959)
myositis by
Curtis
Preliminary
demonstrated in the
with carcinoma of the breast and dermatomyositis to an aqueous extract of her own tumour, and Curtis et al. (1961) in a similar case showed that the antibodies were of a circulatory type. The association of Hashimoto’s disease in our case is perhaps of interest; for of the many diseases thought to be autoimmune in origin, Hashimoto’s thyroiditis is the one woman
whose credentials
beyond reproach. Although we cannot trace a previous report linking Hashimoto’s disease with carcinoma and dermatomyositis or a neuromyopathy, Brain and Henson (1958) note that 3 of the 15 women
are
in their series of
cases
of carcinomatous
myopathy had myxoedema. These patients
were
neuro-
mostly
under observation before there were autoimmune tests for thyroid antibodies and before it was realised that Hashimoto’s disease often ended in myxoedema. The thyroid of 1 of these patients is described in a necropsy report (case 1) by Henson et al. (1954), and the focal lymphocytic infiltration, degenerating acini, and extensive fibrous replacement are consistent with autoimmune thyroiditis. It is clear, however, that if thyroiditis had been the cause of the myxcedema it must have developed long before the carcinomatous neuropathy, since the patient had been taking thyroid for several years. Case 2 in the same series was a woman with a bronchial carcinoma, and although she was not myxcedematous, the thyroid gland at necropsy showed degeneration of acini and focal lymphatic infiltration. In another series, of patients with carcinomatous neuropathy reported by Brain et al. (1951), there was 1 woman (case 1) who was not myxoedematous at the time of her death, but her thyroid gland showed lymphocytic infiltration to a remarkable degree " though less pronounced than in Riedel’s struma. These findings suggest Hashimoto’s disease, and it seems not unlikely that this may have been the cause of the myxoedema in the other two women reported by Brain and Henson (1958). Hall et al. (1962) suggested that the tendency to form thyroid antibodies may be an inherited characteristic; and if the association between Hashimoto’s disease and carcinomatous neuropathy is confirmed and is found to be greater than could be explained by chance, it would lend support to the speculation that carcinomatous neuromyopathy is related to a constitutional reaction of some patients to a neoplasm rather than to any toxic or metabolic effects of the tumour. Nevertheless, Brain and Henson (1958) rightly warn against any facile interpretation of carcinomatous neuromyopathy, and the difference in sex incidence between this condition and Hashimoto’s disease makes the suggested association of doubtful significance, and offers no explanation for the higher incidence of neuromyopathy in certain tumours. "
histological
Communications
OBSERVATIONS ON THE NATURE, DISTRIBUTION, AND SIGNIFICANCE OF
CEPHALOSPORINASE cephalosporins are a series of antibiotics with a common nucleus containing a fused P-lactam dihydrothiazine ring very similar to the fused p-lactam thiazolidine ring of the nucleus of the penicillins. The general properties of these two groups of antibiotics are very much the same, and their mode of action is probably identical. The cephalosporins have recently been completely reviewed by Abraham.1 The promising features which may be exploited for clinical purposes include a suggested absence of cross-allergenicity with the penicillins,2 a broader gram-negative spectrum,3 and resistance to staphylococcal penicillinase.4 Penicillin is almost unique among antibiotics in clinical use in that resistance of bacteria to it is very often, and in some species practically always, mediated by enzymic destruction of the drug. Of chief clinical interest has been the P-lactamase, because of its close association with the resistance of staphylococci. Penicillin acylases, which split off the side-chain but do not affect the nucleus, also exist and contribute to resistance, particularly in the gram-negative species.5 We report here the production (by a species of aerobacter) of an enzyme, apparently a P-lactamase, which is highly active against cephalosporin C (ex-amino adipyl cephalosporin) but which has little if any such action against penicillin G. We have also observed that enzymes degrading the cephalosporins, whether j3-lactamase(s) or otherwise, are widely distributed within the enterobacteriacex, and that in some species their presence is remarkably constant. In general, the most resistant species are among those which produce these enzymes, whereas in the sensitive species we have not been able to THE
detect their presence. EXPERIMENTAL STUDIES
A strain of Enterobacter cloacae (Aerobacter subgroup A) isolated from a blood-culture was found to be tolerant to approximately 10,000 flg. per ml. of cephalosporin C. Initial investigation revealed that it produced a filterable enzyme which rapidly inactivated the antibiotic. 61 An enzyme concentrate (nitrogen content, 13-2%) was prepared from the cell-free culture supernatant by precipitation with saturated ammonium sulphate. This preparation was tested against the following substrates: cephalosporin C, deacetyl cephalosporin C, cephalosporin G (benzyl cephaloAbraham, E. P. Pharmacol. Rev. 1962, 14, 473. Stewart, G. T. Lancet, 1962, i, 509. Chauvette, R. R., Flynn, E. H., Jackson, B. G., Lavagnino, E. R., Morin, R. B., Mueller, R. A., Pioch, R. P., Roeske, R. W., Ryan, C. W., Spencer, J. I., Van Heyningen, E. J. Amer. chem. Soc. 1962, 84, 3401. 4. Jago, M., Heatley, N. G. Brit. J. Pharmacol. 1961, 16, 170. 5. English, A. R., McBride, T. J., Huang, H. T. Proc. Soc. exp. Biol., N.Y. 1960, 104, 547. 6. Fleming, P. C. Canad. J. publ. Hlth, 1963, 54, 47 (abstract). 7. Fleming, P. C., Goldner, M. Fed. Proc. 1963, 22, 206 (abstract no. 274).
1. 2. 3.
Summary A
of 52 had Hashimoto’s disease, dermatomyositis, and carcinoma. Dermatomyositis and carcinomatous neuromyopathy resemble each other in many ways. An association between Hashimoto’s disease and carcinomatous neuromyopathy is suggested and discussed in relation to theories of the nature of the neuromyopathy. woman
W. R.
(1963) Lancet, i, 179. Daniel, P. M., Greenfield, J. G. (1951) J. Neurol. Psychiat. 14, 59. Henson, R. A. (1958) Lancet, ii, 971. Croft, P. B., Wilkinson, M. (1963) ibid. i, 184. Curtis, A. C., Blaylock, H. C., Harrell, E. R. (1952) J. Amer. med. Ass. 150, 844. Heckaman, J. H., Wheeler, A. H. (1961) ibid. 178, 571. Grace, J. T., Dao, T. L. (1959) Cancer, 12, 648. Hall, R., Saxena, K. M., Owen, S. A. (1962) Lancet, ii, 1291. Henson, R. A., Russell, D. S., Wilkinson, M. (1954) Brain, 77, 82. Makari, J. G. (1958) Brit. med. J. ii, 359.
Brain, —
—
—
1400 and penicillin G. A method modified from those of Perret and Tucker 9 was used to ascertain the number of iodine equivalents per mole consumed after the complete alkaline and enzymic hydrolysis of the antibiotics (see table), and to assay the activity of the enzyme concentrate. Infra-redabsorption studies on the lyophilised incubation mixtures were performed both by the KBr-pellet and by the Nujol-mull methods to observe changes in the &bgr;-lactam structure of the
sporin),
8
’
antibiotics. The criteria for identification of enterobacteriaceae were as described by Edwards and Ewing.1O In the penicillins, hydrolysis of the p-lactam ring, either by dilute alkali or by enzyme (p-lactamase) activity, is known to be accompanied by an uptake of 7-9 equivalents of iodine The complete hydrolysis of penicillin G by the aerobacter-enzyme concentrate is also associated with a similar iodine uptake (see table), and this suggests that the penicillinase activity detected by this means is of the lactamase type. The P-lactam ring of cephalosporin C is probably also opened by dilute alkaline hydrolysis,12 and an iodine uptake of 4 equivalents has been reported for this reaction." We confirm this finding and note similar values for deacetyl cephalosporin C and for cephalosporin G. As the results of the enzymic and alkaline hydrolysis of the cephalosporins show such close agreement, it seems likely that the cephalosporinase activity of the concentrate, detected by this means, is also of the p-lactam type. Deacetylation of the acetoxyl side-chain, however, which IODINE
EQUIVALENTS
"
DISTRIBUTION OF
CEPHALOSPORINASES
"
16 was
The membrane method of Knox and Smith modified study the distribution of cephalosporinases and penicillinases in over a thousand strains of enterobacteriacese and associated organisms. This technique involves whole living organisms and detects cell-bound as well as filterable enzyme activity. As used here, it determines only the ability of the organism to produce acid degradation products from the substrate used. It is important to appreciate, therefore, that the terms cephalosporinase and penicillinase used below refer not only to cephalosporin and penicillin &bgr;-Iactamase activity but also to cephalosporin and penicillin acylase and to cephalosporinesterase (deacetylating) activity should these be present. Any of these enzymes may occur singly or in combination.
to
Technique Single colonies of the organism under test were grown overnight, suitably spaced on the surface of a ’Millipore’ membrane laid on a nutrient-agar plate. The membrane was then removed and placed on a Whatman no. 1 filterpaper saturated with a neutralised solution containing cephalosporin C (25,000 ij!.g. per ml.) or penicillin G (25,000 units per ml.), and 2% Andrade’s indicator. Membrane and filterpaper were then incubated at a suitable temperature and examined from time to time for the appearance of a red colour in or about the colony. A control omitting the substrate and using Andrade’s indicator alone was always included. All membranes were observed for six hours. Strong positive reactions showed up within twenty minutes. The acid reaction produced from cephalosporin C, unlike that from penicillin G, is apparently not stable as the colour usually fades within an hour of its appearance. RESULTS
The
organisms placed in one of three groups to their according ability to form acid from cephalosporin C by the membrane technique:
*
Except for deacetyl cephalosporin C, alkaline hydrolysis consumed more iodine than did enzymic hydrolysis.
a
little
is found in the cephalosporins but not in the penicillins, is not excluded. If present here, it does not account for any significant iodine absorption since the iodine equivalents are the same after enzymic hydrolysis of cephalosporin C and deacetyl cephalosporin C (see table).
Infra-red Absorption Studies Direct evidence that the p-lactam ring is opened during enzymic hydrolysis of penicillin G and cephalosporin C was provided by observing the disappearance of the infra-red at 5-62 !L after incubation with the enzyme This band is considered to be characteristic of a part of the intact fused &bgr;-Iactam ring.14 Specific Activities The values of the specific P-lactamase activity of the enzyme concentrate for the different substrates, measured iodometrically, and expressed as microgrammes of antibiotic hydrolysed per minute per milligramme dry weight of concentrate, are:
absorption band concentrate.
were
Group 1.-geneva and species, strains of which were consistently positive: Aerobacter (subgroups A, B, and C), serratia, hafnia, Proteus morganii, and Pseudomonas aeruginosa. Group 11.-Genera and species, strains of which were consistently negative: Salmonella, klebsiella (all species), Pr. mirabilis.
Group I11.-Genera and species, some strains of which were positive and others negative: Pr. vulgaris, Pr. rettgeri, Escherichia coli, Shigella sonnei, and some other Shigella species. Relationship of Cephalosporinase Production to Resistance Group I.-These strains were usually tolerant to several hundred or thousands of microgrammes per millilitre. Group 77.—These were usually sensitive to 5-50 g. per ml. Group 777.—In some species such as Sh. sonnei, cephalosporinase production was obviously related to resistance. In other species, such as Pr. vulgaris which is usually highly resistant, this resistance could not be correlated with ability to produce cephalosporinase. Relationship of Penicillinase to Cephalosporinase Production As determined by the membrane technique, there was no clear association between cephalosporinase and penicillinase activity. Organisms were isolated which showed penicillinase or cephalosporinase activity only, both, or neither. DISCUSSION
The activity of the enzyme concentrate against cephalosporin C very greatly exceeds its activity against penicillin G. We have not yet determined whether both these activities are due to the same or separate enzymes. Perret, C. J. Nature, Lond. 1954, 174, 1012. Tucker, R. G. ibid. 1934, 173, 85. 10. Edwards, P. R., Ewing, W. H. Identification of Enterobacteriaceæ. 8. 9.
Minneapolis, 1962. 11. Alicino, J. F. Industr. Engng Chem. (Anal.) 1946, 18, 619. 12. Crompton, B., Jago, M., Crawford, K., Newton, G. G. F., Abraham, 13. 14.
E. P. Biochem. J. 1962, 83, 52. Alicino, J. F. Anal. Chem. 1961, 33, 648. Abraham, E. P., Newton, G. G. F. Biochem. J. 1956, 62, 651.
Abraham and Newton,14 and later Crompton et al.,12 reported that culture filtrates of Bacillus cereus showed cephalosporinase (p-lactamase) in addition to penicillinase ((3-lactamase) activity. They noted that the activity of these filtrates was low against cephalosporin C, deacetyl cephalosporin C, and cephalosporin G, compared to penicillin G. Moreover, purified penicillinase prepared from these filtrates showed even less relative cephalosporinase activity. In contradistinction, both our aerobacter filtrate and the enzyme preparation derived from it were highly active against cephalosporin C, but they 15.
Knox, R., Smith, J. T. Nature, Lond. 1961, 191, 926.
1401 or little activity, respectively, against penicillin small amount of penicillinase activity present in The G. the concentrate could be derived from autolysed bacterial cells since it was shown by the membrane technique that this strain gave both strong penicillinase and cephalosporinase reactions. The varying activity of the enzyme against different cephalosporins suggests the possible development of
showed no
" cephalosporinase-resistant " cephalosporins. With the use of cephalosporins clinically,6 16 knowledge of the existence and distribution of inactivating enzymes is of obvious importance. The regular presence or absence of cephalosporinase in certain species may also be of "
"
taxonomic value. The
The Research Institute, Hospital for Sick Children, Toronto, Canada
P. C. FLEMING M.B.
Lond.
M. GOLDNER PH.D.
Connaught Medical Research Laboratories, University of Toronto
Montreal
D. G. GLASS B.SC.
McMaster
SPLENIC-TO-FEMORAL AND AXILLARY-TO-FEMORAL BYPASS GRAFTS IN DIFFUSE ATHEROSCLEROTIC OCCLUSIVE DISEASE ALTHOUGH restoration of arterial flow in atherosclerotic occlusive disease has now become standardised and
relatively simple, the salvage of legs in elderly or poor-risk patients with impending or overt gangrene still poses a difficult problem. In about half the cases, combined aorto-iliac and femoro-popliteal occlusions are found, and there is significant disease of the popliteal artery and its branches in almost 40%.1’ 18 In such cases satisfactory revascularisation of the legs entails a major procedurenamely, aorta-to-femoral-to-popliteal bypass graft 19 or aorto-iliac endarterectomy combined with femoral-toSociety for Microbiology. Antimicrobial Agents and Chemotherapy. Ann Arbor, 1962 (in the press). Louw, J. H., Roberts, W. M. S. Afr. med. J. 1961, 35, 346, 367, 385. Morris, G. C., Jr., Wheeler, C. G., Crawford, E. S., Cooley, D. A., De Bakey, M. E. Surgery, 1962, 51, 50. Morris, G. C., Jr., Edwards, W., Cooley, D. A., Crawford, E. S., De Bakey, M. E. Arch. Surg. 1961, 82, 32.
16. American 17. 18. 19.
These operations may, however, be unwise because of the poor condition of the patient, or impossible because of the extent of the disease. The surgeon, faced with the risk of a probable " amputation for mortification ", may be obliged to perform a compromise operation which will provide satisfactory, although not ideal, revascularisation. This can be achieved by limiting or modifying either the distal or the proximal extent of.the standard reconstructive procedure and combining it with sympathectomy when possible." 8-23
popliteal bypass graftsY
DISTAL LIMITATION
The value of simply restoring an adequate blood-flow to the profunda femoris artery in patients with combined aortò-iliac and femoro-popliteal occlusions has been well established. We have achieved this mainly by aorto-iliac endarterectomy with 70% success 17; others have claimed even better results with aorta-to-femoral bypass grafts combined with sympathectomy.120 The profunda femoris artery almost always remains remarkably free of atherosclerosis, and even if the common femoral artery appears completely occluded on arteriography, the patient should not be denied the benefit of this type of operation, with or without concomitant sympathectomy. 1-9 20 PROXIMAL LIMITATION
patients a good pulsatile flow is present in the aorta above the occlusion; hence anastomosis of a graft to the aorta or aorto-iliac endarterectomy is usually possible. But sometimes, because of extensive calcific or ulcerating disease, " irremovable aneurysms ", or fibrosis due to failure of previous vascular operations, these procedures are impossible or unduly hazardous. Moreover, operations on the aorta and iliac arteries have a high mortality-rate and morbidity-rate.24 A more suitable or accessible artery for the feeding vessel " may have to be used. The splenic artery has been used by several workers,25 and we have used it in 4 patients with remarkable success In
most
"
20. 21. 22. 23. 24. 25.
Leeds, F. H., Gilfillan, R. S. ibid. p. 25. McCaughan, J. J., Jr., Kahn, S. F. Ann. Surg. 1960, 151, Vetto, R. M. Surgery, 1962, 52, 342. Warren, R. Arch. Surg. 1956, 72, 57. Cockett, F. B., Maurice, B. A. Brit. med. J. 1962, i, 353. Freeman, N. E., Leeds, F. H. Surgery, 1953, 34, 1021.
BYPASS OPERATIONS IN VASCULAR INSUFFICIENCY
26.