Six cephalosporins (currently available for clinical use or undergoing clinical trials) were tested by tube dilution method against high inoculum (107 colony forming units) of 37 strains of penicillin resistant Staphylococcus aureus. 3.1 μg/ml of cephalothin inhibited 100% of strains while higher concentrations of cefoxitin, cephapirin, cefamandole, cephalexin and cephradine were required. The percent of strains with minimal inhibitory concentration higher than 3.1 μg/ml ranged between 10.8% (cefoxitin) and 100% (cephradine). These findings support the observation that cephalothin is less susceptible to inactivation than the other cephalosporins. Although the exact clinical implication of these findings has not been established, cephalothin might be the cephalosporin of choice for treatment of severe infections caused by penicillin resistant Staphylococcus aureus

del Busto, Ramon

Pohlod, Donald

Quinn, Edward L.

Suarez, Andres

Henry Ford Health System Scholarly Commons

Henry Ford Hospital Medical Journal Volume 24 Number 4 Article 2 12-1976 Susceptibility at high inoculum of penicillin-resistant Staphylococcus aureus to six cephalosporins Andres Suarez Ramon del Busto Edward L. Quinn Donald Pohlod Follow this and additional works at: https://scholarlycommons.henryford.com/hfhmedjournal  Part of the Life Sciences Commons, Medical Specialties Commons, and the Public Health Commons Recommended Citation Suarez, Andres; del Busto, Ramon; Quinn, Edward L.; and Pohlod, Donald (1976) "Susceptibility at high inoculum of penicillin-resistant Staphylococcus aureus to six cephalosporins," Henry Ford Hospital Medical Journal : Vol. 24 : No. 4 , 187-190. Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol24/iss4/2 This Article is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Henry Ford Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons. Henry Ford Hosp Med Journal Vol 24, N o 4, 1976 Susceptibility at high inoculum of penicillin-resistant Staphylococcus aureus to six cephalosporins Andres Suarez, M D ; Ramon del Busto, M D ; Edward L. Q u i n n , M D , and Dona ld Poh lod, M D * Six cephalosporins (currently available for clinical use or undergoing clinical trials) were tested by tube dilution method against high inoculum ( W colony forming units) of 37 strains of pen i c i l l i n resistant Staph-ylococcus aureus. 3./ p g / m l of cephalothin inhibited 100% of strains while higher con-centrations of cefoxitin, cephapirin, cefa-mandole, cephalexin and cephradine were required. The percent of strains with mini-mal inhibitory concentration higher than 3.1 p g / m l ranged between 10.8% (cefoxitin) and 100% (cephradine). These findings sup-port the observation that cephalothin is less susceptible to inacUvation than the other cephalosporins. Although the exact clinical implication of these findings has not been establ ished, cephaloth in might be the cephalosporin of choice for treatment of severe infections caused by penicill in resist-ant Staphylococcus aureus. * Division of Infectious Diseases This study was supported by grants from the Michigan Heart Association and the McGraw Foundation. Address reprint requests to authors at Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, Ml 48202 THE usefulness of the cephalosporins as alternate agents in the treatment of staph-ylococcal endocarditis is well established.'-'' However, several cases have been reported of clinical and bacteriological failure when treatingStaphy/ococcus aureus endocarditis with cephaloridine or cefazolin despite the in vitro susceptibility ofthe staphylococci to these antibiotics by conventional tests. Bur-gess and Evans' reported a patient in whom bacteremia persisted after five days of treat-mentwith cephaloridine despite presence of 12.5 p g / m l of the antibiotic at its lowest concentration. Further study ofthe organism showed that although the minimal inhibitory concentration (MIC) of cephaloridine when tested w i t h a small inocu lum was 0.12 /xg/ml , it rose to 4 |U.g/ml when a high inoculum was used. Other in vitro studies',' have indicated that the susceptibility of pen-icillin resistantS. aureus to cephaloridine is markedly dependent on the size ofthe inoc-ulum used for the test. In some cases a greater than 100 fold increase in the MIC was observed when the inoculum was increased from 10" to 10' colony forming units/ml while the MIC of cephalothin showed only a 10 fold increase as maximum. It was also shown that inactivation by Beta lactamase was responsible for these findings. Quinn et aP reported a series of ten patients with penicillin resistant S. aureus endocarditis who were treated with intra-187 Suarez, del Busto, Qu inn , Pohlod muscular cefazolin. The MIC's of all ten strains, as determined with standard inocu-lum of approximately 10^ colony forming u n i t s / m l , ranged between 0.2 and 0.8 ; i g /m l of cefazolin. One patient failed to respond to treatment and had positive blood cultures while cefazolin was being given. Further study of the organism infecting this patient showed that the MIC increased to 15 p g / m l with a high inoculum (10' colony forming units/ml). This marked inoculum effect was not observed for strains of staph-y lococc i f rom patients who responded favorably to cefazolin. Itwas also shown that the strain that showed the marked inoculum effect (or was "resistant" when tested at high inoculumjwasableto inactivate cefazolin in vitro. Based on the above mentioned studies, it is clear that the activity of different cephalosporins against penicillin resistantS. aureus is related to their susceptibility to inactivation by a Beta lactamase. This is reflected by higher MIC's at high inoculum and may be associated with therapeutic failure. The present study was designed to determine the frequency with which high concentrations of cephalosporins (currently available and under clinical trial) will be required to inhibit penicillin-resistant strains of S. aureus. Material and methods We studied 37 strains of S. aureus isolated from patients at the Henry Ford Hospital. Except for their resistance to penicillin C and their suscept ib i l i ty to meth ic i l l i n , these strains were unselected. Susceptibilities to the different cephalosporins were studied by determining the MIC using the standard macrotube dilution method.'" Standard an-tibiotic powders were provided by the fol-lowing laboratories: cephradine by Smith, Kline and French Laboratories, Philadelphia, Pennsylvania; cefoxitin by Merck, Sharp and Dohme, Rahyway, New Jersey; sodium cephapirin by Bristol Laboratories, Syracuse, New York; and sodium cepha lo th in , cephalexin monohydrate and cefamandole l i th ium by Eli Li l ly and Company, Ind i -anapolis, Indiana. The culture media used was trypticase soy broth (BBL). The inocu-lum consisted of 1 ml ofa 1 0 " ' di lut ionof an 18-hour culture o f the strain being studied. Bacterial plate counts using antibiotic free media were performed for all strains in order to determine the exact size of the inoculum which was later found to range from 0.4x10' to 3x10' colony forming units. Each strain was simultaneously tested against all antibi-otics. MIC was defined as the minimum concentration of antibiotic necessary to in-hibit growth after 18 hours of incubation. Results Our results are summarized in Figure 1. It can be seen that only 3.1 pg,/ml or less of cephalothin was required to inhibit 100% of strains tested. On the other hand, higher concentrations of cefoxitin, cephapirin, cefamandole, cephalexin and cephradine, in that order, were required for the same purpose. In Table 1 the population of bacteria has been divided in two groups: those strains inhibited by 3.1 p g / m l or less ofthe various antibiotics and those strains inhibited by 6.2 p g / m l or more. We chose 3.1 p g / m l as the dividing point because when tested with conventional inoculum (10^ colony forming units/ml) this concentration of cephalothin, ce fox i t in , cefamandole, cefazol in and cephalexin inhibited growth of 100% of strains of penicillin resistant S. aureus iso-lated from patients with endocarditis and tested in our laboratory. The table shows that all strains were inhibited by 3.1 p g / m l of cephalothin whereas 6.2 /Ag/ ml or more was required to inhibit 10.8% of strains with cefoxitin, 24.3% of strains with cephapirin, 40.5% of strains with cefamandole, 72.9% of strains with cephalexin and 100% of strains with cephradine. Discussion When the in vitro activity against pen-icillin resistant Staphylococcus aureus is 188 Susceptibility of S. aureus to six cephalosporins 100 -I (100%) m X z < a: (O Ll. o LLJ > zs o CEPHALOTHIN • • CEFOXITIN o o CEPHAPIRIN *• •* CEPHALEXIN A CEFAMANDOLE • • CEPHRADINE .-"(0%) —. r-/y^ 0.048 0097 0195 039 0.78 1.56 3.1 6.2 12.5 25 50 >50 ANTIBIOTIC CONCENTRATION ^ g / m l Figure 1 Cumulative percent of strains of Staphylococcus aureus inhibited at increasing concentrations of six cephalosporin antibiotics. Percent of strains inhibited by 3.1 j i ig /ml or less indicated in parenthesis. Studied by the tube d i l u t i on me thod using a h igh i n o c u l u m (10' co lony f o rm ing un i t s / ml) , our results indicate that a lower c o n -cent ra t ion o f cepha lo th in is requi red to in -hib i t the g row th o f 100% of strains. For at least 10 .8% and as m u c h as 100% of strains, h i g h e r c o n c e n t r a t i o n s o f t h e o t h e r c e p h a l o s p o r i n s are r e q u i r e d to i n h i b i t g row th . The de f in i t i on o f suscept ib i l i ty or resist-ance o f an organism to a given ant ib io t ic impl ies not on l y results of In v i t ro suscep-t ib i l i t y testing but the cor re la t ion of these results w i t h those of pharmacok ine t i c s tud-ies and c l i n i ca l tr ials w i t h the d rug in ques-t i on . Therefore, the conc lus ion that a given ant ib io t ic is better o r worse than the other based on l y on data o f their act iv i ty ;'n v i t ro is not va l id . However , the design of our study and the interpretat ion of its results are based on c l i n i ca l and bacter io log ica l ev idence in -d i c a t i n g t h a t f a i l u r e o f acepha lospo r i n in the t reatment o f endocard i t i s due to pen ic i l l i n TABLE I Cephalothin Cefoxitin Cephapirin Cefamandole Cephalexin Cephradine MIC* No. % No. % No. % No. % No. % No. % 3.1 /xg/ml or less 37 100% 33 89.2% 28 75.7% 22 59.5% 10 271% 0 0% 6.2 /xg/ml or more 0 0% 4 10.8% 9 24.3% 15 40.5% 27 72.9% 37 100% * MIC — minimal inhibitory concentration determined wi th high inoculum (10'colony forming uni ts/ml) Number and percent of strains o i Staphylococcus aureus inhibited by 3.1 /Ag/ml or less and 6.2 / t g / m l or more of the six cephalosporins tested. 189 Suarez, del Busto, Quinn, Pohlod resistantS. aureus migh t be related to high MIC of the ant ib io t ic w h e n tested at h igh i n o c u l u m . This reflects the suscept ib i l i ty of the ant ib io t ic to dest ruct ion by Beta lac-tamase produced by the organisms. ' - ' ' Our results support the observation that cephalothin is less susceptible to inactiva-tion by Beta lactamase than the other cephalosporins."-' This observation has been confirmed recently by studying the inactivation of eight cephalosporin antibiot-ics in broth culture of growing S. aureus." p e r h a p s o t h e r t y p e s o f seve re i n f e c t i o n c a u s e d by p e n i c i l l i n r e s i s t a n t S . a u r e u s , cepha lo th in w o u l d be the d rug of cho ice . T h e d e c r e a s e d e f f e c t i v e n e s s o f o t h e r cepha lospor ins for severe infect ions may depend on their suscept ib i l i ty to inact ivat ion by the strain o f s taphylococcus be ing treat-ed . Such suscept ib i l i ty to inact ivat ion may be presumed to be s igni f icant if the M I C using an i n o c u l u m of 1 0 ' co l ony f o rm ing u n i t s / m l is marked ly higher than the M I C using a standard i nocu lum. The exact c l i n i ca l imp l ica t ions o f our f indings are not w e l l establ ished. However , a t the present state o f know ledge , w e tend to agree w i t h others'^- '^ that, if a cepha lospor in is used f o r t h e t reatment o f endocard i t is , and A c k n o w l e d g m e n t W e thank Margaret Somerv i l le for ski l l fu l techn ica l assistance. References 1. Benner Ej: Cepha lospor in an t ib io t ics : 8 Therapeutic dimensions and future. Postgrad Med 1 47 (suppl):135-142, 1971 2. Herrell WE, Balows A and Becker J: Some e x p e r i m e n t a l and c l i n i c a l s tud ies on 9 c e p h a l o t h i n . C/ /n P h a r m a c o l Ther 4 : 709-719, 1963 3. Quinn EL, Pohlod D, Madhavan T, Burch K, 10 Fisher E and Cox, F: Clinical experiences with Cefazolin and other cephalosporins in bac-terial endocarditis. / Infect Dis 128 (Suppl): 386-391, 1973 4. Rahal Jj, Meyers BR, and Weinstein L: Treat- n . men t o f b a c t e r i a l e n d o c a r d i t i s w i t h cephalothin. N Eng! j Med 279:1305-1309, 1968 5. Smith I M : Cepha lospor in therapy of sta-phylococcal infections in adults. Postgrad 12. M e d ; 47 (Suppl):78-87, 1971 6. Zabransky RJ, Gardener MA and Geraci JE: Cephalexin: Preliminary in vitro studies of a new orally administered cephalosporin and 13 report of a case of endocarditis cured with cephalexin. Mayo C//n Proc 44:876-885, 1969 7. Burgess HA and Evans Rj ; Failure of Cephaloridine in a case of Staphylococcal endocarditis. Br Med j 2:1244,1966 Benner EJ, Bennett JV, Brodie JL and Kirby W M M : Inact ivat ion of Cephalo th in and Cephaloridine by Staphylococcus aureus, j Bacteriol 90:1599-1604,1965 Ridley M and Phillips I: Relative instability of cepha lor id ine to s taphylococcal pen-icillinase. Nature 206:1076-1078, 1965 Washington JA and Barry AL: Dilution tests procedures p. 414. In E. H. Leunette, E. H. Spaulding and 1. P. Truant (ed.). Manual of Clinical Microbiology, 2nd ed. American So-ciety for Microbiology, Washington, DC, 1974 Sabath LD, Garner C, Wilcox C and Finland M: Effect of inoculum and of Beta lactamase on the anti-staphylococcal activity of thirteen penicillins and cephalosporins. AnUmicrob Agents Chemother 8:344-349,1975 Fong IW, Engelking ER and Kirby W M M : Relative inactivation by Staphylococcus au-reus of eight cephalosporin antibiotics. Anti-microb Agents Chemother 9:939-944,1976 Rogers DE and Turck M : Staphylococcal infections, p. 776. In M. M. Wintrobe, G. W. Thorn, R. D. Adams, E. Braunwald, K. J. Isselbacher and R. G. Petersdorf, (ed), Harri-son's Principles of Internal Medicine, 7th ed. McGraw-Hil l Book Co, New York, 1974 190 

Susceptibility at high inoculum of penicillin-resistant Staphylococcus aureus to six cephalosporins

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