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Department of Infectious Diseases, University of Gdteborg, Ostra Sjukhuset, S-41685 Gdteborg, Sweden References Bailey, R. R-, Roberts, A. P., Gower, P. E. & de Wardener, E. H. Prevention of urinary tract infection with low dosage nitrofurantoin. Lancet ii: 1112-14(1971). Brumfitt, W. & Hamilton-Miller, J. M. T. Longterm treatment with trimethoprim. Lancet ii: 210 (1978). Brumfitt, W. & Purcell, R. Double-blind trial to compare ampicillin, cephalexin, co-trimoxazole, and trimethoprim in treatment of urinary infection. British Medical Journal ii: 673-6 (1972). Cartel, W. R-, Chamberlain, D. A., Fry, I. K., McSherry, M. A., Broughton, C. & O'Grady, F. Long-term control of bacteriuria with trimethoprim-sulphonamide. British Medical Journal i: 377-9(1971).

Freeman, R. B., Bromer. L., Brancatao, F., Cohen, S. I., Garfield, C. R., Griep, R. J., Hinman, E. J., Richardson, J. A., Thurm, R. H., Winer. C. & Smith, W. M. Prevention of recurrent bacteriuria with continuous chemotherapy. Annals of Internal Medicine 69: 655-60 (1968). Goodman, L. S. & Gilman, A. (ed.) The pharmacological basis of therapeutics 5 ed. New York (1975). Hoigne, R., Barhtolome, P. J. & Muller, U. Bactrim Roche, ein Kombinations-praparat von Sulfamethoxazol und Trimethoprim. Schweitzischer Medizinischer Wochenschrift 99: 1511-15 (1969). Kasanen, A., Toivanen, P., Sourander, L., Kaarsalo, E. & Aantaa, S. Trimethoprim in the treatment and long-term control of urinary tract infection. Scandinavian Journal ofInfectious Diseases 6: 91-6 (1974a). Kasanen, A., Kaarsalo, E., Hiltunen, R. & Soini, V. Comparison of long-term low-dosage nitrofurantoin, methenamine hippurate, trimethoprim and trimethoprim-sulphamethozaxole on the control of recurrent urinary tract infection. Annals of Clinical Research 6: 285-9 (19746). Larsson, S., Cronbcrg, S., Dennerbert, T. & Ohlsson, N. M. Pulmonary reactions to nitrofurantoin. Scandinavian Journal of Respiratory Diseases 54: 103-10 (1973). Iindberg, J. Clinical and pathogenetic aspects on chronic active hepatitis. Scandinavian Journal of Infectious Diseases Suppl. 12 (1978).

Inactivatlon of cephalosporins in blood cultures and mixed assays with a commercially available enterobacter {3-bctamase Sir, In our report on the comparative activity of several P-lactamases, the unavailability of a commercial preparation of the P99-type cephalosporinase was noted with regret (Selwyn, 1977). This form of enzyme is required to inactivate fHactamase resistant cephalosporins and cephamycins in blood cultures from patients on treatment. It is also invaluable when a patient is simultaneously receiving an aminoglycoside which requires to be assayed in the serum. For such mixed serum assays the use of Klebsiella edwardsii var. at Ian toe, NCTC 10896, has been recommended as the test organism (Shanson & Hince, 1977; Holt & Reeves, 1978); but unfortunately this strain is inhibited in broth by five out of twelve cephalosporins tested at a concentration of 10 ug/ml or less (Table I). Although binding to serum proteins reduces the activity of several of the antibiotics, including cefoxitin (Table I), this may have a minimal effect under the conditions of the assay, since the medium itself contains no protein.

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occurred in only one of 13 patients with a total period of treatment of 92 months. This means a recurrence rate of 11 % per month of prophylaxis. Both recurrence rates are low and even if the patient groups are rather small, the total periods of medication are of considerable length. Side effects appearing during co-trimoxazone treatment have as a rule been suggested to be attributed to the sulphonamide component. However, trimethoprim may also give adverse reactions of the hypersensitivity type. Kasanen et al. (19746) compared side effects of co-trimoxazole, nitrofurantoin and trimethoprim. The long-term prophylaxis was interrupted because of side effects in 3-3% (nitrofurantoin), 3-2% (co-trimoxazole) and 1-5 % (trimethoprim). In the present study the long-term treatment was interrupted because of side effects in one patient (6-7 %), but more extensive studies have to be performed before the true incidence of adverse reactions during short- and long-term trimethoprim medication has been fully elucidated. In view of the present findings the recent suggestion by Brumfitt & Hamilton-Miller (1978) of a multi-center trial on long-term, low-dosage, trimethoprim treatment of urinary tract infections seems worth attempting. Trimethoprim may prove to be a useful drug provided that any increase in the occurrence or urinary pathogens resistant to the drug, are carefully monitored. STEN IWARSON GUNILLA LIDIN-JANSON

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Plate 1. Destruction of cephalosporins by Enterobacter cloacae P-lactamase (Miles); (a) 1-2 i.u./linear cm (b) 12 i.u./linear cm. Discs containing 10 pg of antibiotic are from above downwards: Qcft) cephalondine (CP), cephalothin (CT), cephalexin (CX), cephradine (CH), and cefadroxil (CF); (right) cephazoUn (CZ), cefamandole (CD), cefuroxime (CM), cefoxitin (CN), cephacetrile (CR), and cephapirin (CI).

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319

Table I.

Antibiotic

(0*

00*

(iii)*

10

100

70 50 8 8 10 25 25 30 4 5 30 40

>100 >100 15 15 30 60 >100 100 20 15 80 80

>100 >100 15 15 25 50 80 60 15 10 60 80

0-004 0-004 0-009 0-01 0-009 0-02 0-004 0-01 11 2-2 (K)l 0-004

0-15 0-08 0-2 1-8 0-2 1-8 0-08 2-5

16

27 0-8 0-08

•ug/ml in serial dilution test (close series) in: (i) Oxoid Sensitest Broth alone; (if) 94% pooled human serum in broth; (iii) 4% human serum albumin in broth.

Cefuroxime poses no problems in mixed assays because of its great vulnerability to Bacillus cereus type II enzyme, as contained in the Whatman 'P-lactamase Broad Spectrum Mixture'; but this enzyme has an inadequate effect on cefoxitin and cephradine, and is only moderately active against several other cephalosporins (Selwyn, 1977; Imambaccus et al., 1978). A preparation of P99-type P-lactamase (Class la) obtained by ultrasonication of Enterobacter cloacae is fortunately now available commercially ('Cephalosporinase', Miles Laboratories, Stoke Poges, England). It is supplied frozen in 5 ml ampoules containing approximately 2000 international units (i.u.) of enzyme per ml. Batches examined have ranged in potency from 1190 i.u. to 2900 i.u./ml. There is no significant loss of activity when the enzyme is stored for six months at -20°C. The spectrum of activity of the enzyme is demonstrated in Plate 1, using the solid assay system previously described (Selwyn, 1977). The small zones of inhibition around the cefuroxime and cefoxitin discs remaining after enzyme action represent less than 1 % residual antibiotic. The amounts of the enzyme preparation required to inactivate the various cephalosporins and cefoxitin present in pooled samples of human serum at a concentration of 10 and 100 ug/ml are shown in Table I. In practice, the addition of 0-1 ml of a 1 in 10 dilution of the enzyme preparation (containing approximately 20 i.u.) to 1 ml of patient's

serum will destroy 100 ug of any of the cephalosporins or cefoxitin. A proportionately larger amount of enzyme should be added to blood culture bottles (0-25 ml has been found adequate when 5 ml whole blood is cultured). According to the definition of the international unit, only 1 min is required for complete hydrolysis at 25°C (the old Pollock unit allowed an hour, but a mere second is required according to the S.I. unit definition). It would seem wise, however, to allow the enzyme to act in serum for 10 min at 37°C, mixing thoroughly, before performing an aminoglycoside assay. An equivalent P99-type P-lactamase preparation, as well as a mixture of this enzyme with Escherichia coll (TEM) and B. cereus (569/H/9) P-lactamase, have been available since 1976 from Dr J. Melling, Microbiological Research Establishment, Porton. A mixture of this type has proved ideal for general use against all P-lactam antibiotics (to be reported). The two enterobacter enzyme preparations when used alone have even proved potent enough to allow sterility tests to be performed on oral cephradine, when this has been used in immunodeficient patients who are nursed in ultrastrict isolation (Watson et al., 1977). S. SELWYN M. BAKHTIAR Department of Medical Microbiology, Westminster Medical School, London, England

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Cephaloridine Cephalothin Cephalexin Cephiadine Cefadroxil Cephaloglycin Cephazolin Cefamandole Cefoxitin Cefuroxime Cephacetrile Cephapirin

Amount of Enterobacttr P-lactamase (i.u./ml) required to neutralize antibiotic concentrations (ug/ml)

MIC of Klebsiella ]MCTC 10896

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References ticarcillin are included in the zone size Holt, H. A. & Reeves, D. S. Assays of mixtures of interpretative charts provided as package Antibiotics. In Laboratory Methods in Anti- inserts with standard antimicrobial discs, and microbial Chemotherapy (Reeves, D. S., Phillips,the zone sizes listed for carbenicillin mention I., Williams, J. D. & Wise, R., Edj). Churchill only Pseudomonas aeruginosa, Escherichia coli, Livingstone, Edinburgh, London and New York and the Proteus spp. Therefore, most labora(1978), p. 164. Imambaccus, Y., Sclwyn, S. & Bakhtiar, M. tories use the zone sizes listed for E. coli and Susceptibility of cefamandole, cefoxitin, and Proteus spp. as standard for all Enterocefuroxime to beta-lactaroases. Current Chemo- bacteriaceae. The manufacturer of ticarcillin provides 75 ug discs to diagnostic laboratories therapy 1: 495-7 (1978). Selwyn, S. Susceptibility of penicillins and cephalo- to be used for susceptibility testing. It also sporins to fJ-lactamases assessed by a new test. supplies data regarding acceptable zone sizes Journal of Antimicrobial Chemotherapy 3:for the three previously mentioned organisms. 161-68 (1977). Since zone sizes of 11 mm or less are conShanson, D. C. & Hince, J. C. Factors affecting sidered resistant; 12 to 14 mm are considered plate assay of gentamicin III. Klebsiella assay intermediate; and 15 mm or greater are constrains. Journal of Antimicrobial Chemotherapy sidered susceptible for P. aeruginosa, E. coli, 3: 563-70 (1977). Watson, J. G., Rogers, T. R-, Selwyn, S. & Smith, and the Proteus spp., these zone interpretations R. G. Evaluation of the Vickers-Trexler isolator are also commonly used for K. pneumoniae in children undergoing bone marrow trans- and other Enterobacteriaceae even though the plantation. Archives of Disease in Childhood SI:manufacturer makes no claims for these 563-8 (1977). organisms. In our laboratory all organisms implicated Comparison of disc zone sizes with minimum in disease processes are checked for susceptiinhibitory concentrations using bility to a standard battery of antimicrobial semlsynthetic penicillin agents by the Bauer-Kirby disc method. Sir, Since it has been found that zone size is Many institutions use protocols for initial exquisitely sensitive to inoculum size, one empiric therapy prior to isolation, identifica- technician was assigned to run all of these tion, and subsequent testing for antimicrobial tests, including the minimum inhibitory susceptibility of organisms which cause infec- concentrations (MICs) and the measurement tion. The EORTC International Antimicrobial of zone sizes with calipers in the Bauer-Kirby Therapy Group (1978) reported that a test. The MICs were determined by a modificacombination of carbenicillin or ticarcillin and tion of the semiautomatic microtiter method an aminoglycoside would appear to have the described by MacLowry et al. (1970). Muellerbest ratio of efficacy to toxicity for initial Hinton broth was used rather than trypticase empiric therapy of infection in granulocyto- soy broth and both the Mueller-Hinton broth penic patients with cancer. At the Baltimore and agar for each method were adjusted prior Cancer Research Center (BCRQ, ticarcillin is to autodaving to give a concentration of 20 to administered in conjunction with an amino- 35 mg of magnesium and 75 to 100 mg of glycoside when a cancer patient with less than calcium per liter as proposed by Reller et al. 1000/ml absolute granulocyte count has a fever (1974). Each MIC was repeated twice and of unknown origin. We have observed at each Bauer-Kirby test at least 3 times. BCRC that the clinical laboratory antibiotic When the results of Bauer-Kirby disc susceptibility results may be invalid when susceptibilities to the two semisynthetic standard methods are used to determine the penicillins, ticarcillin, and carbenicillin were susceptibility of Klebsiella pneumonias to compared to MICs, it was discovered that the ticarcillin. Although ticarcillin is not the carbenicillin results correlated well, but there drug of choice for therapy of infections caused were wide variations in the results when by K. pneumonias the package insert which is ticarcillin was tested against K. pneumoniae. distributed with the drug in the United States A comparison of K. pneumoniae Bauer-Kirby specifies that 'Quantitative methods that disc susceptibility and MIC test tesults against require measurement of zone diameters such ticarcillin and carbenicillin are shown on as Bauer-Kirby give the most precise esti- Table I. Of more than 850 bacterial strains mates of antibiotic susceptibility for ticarcillin, isolated in this study, 145 were identified as and a report of susceptible indicates that the K. pneumoniae. One hundred and twenty-two infecting organism is likely to respond to of these K. pneumoniae strains appeared to therapy'. However, no zone size standards for be susceptible or intermediate to ticarcillin by