Eur. J. Clin. Microbiol. Infect. Dis.

centration options for piperacillin-tazobactam. The available human pharmacokinetic data would support the use of either testing option; however, the 8:1 ratio of piperacillin to tazobactam may be preferable given that the clinical formulation contains the two compounds in an 8:1 ratio and the similar pharmacokinetic properties of piperacillin and tazobactam should ensure that the administered ratio is maintained in vivo. Ultimately, the choice of testing option will depend upon the adoption of a uniform approach to the testing of beta-lactamase inhibitor combinations. Until such time, the fixed 8:1 ratio tazobactam combination will suffice for clinical testing of piperacillin-tazobactam combinations.

References 1. Aronoff SC, Jacobs MR, Johenning S, Yamabe' S: Comparative activities of the beta-lactamase inhibitors YTR 830, sodium clavulanate, and sulbactam combined with amoxicillin or ampicillin. Antimicrobiat Agents and Chemotherapy 1984, 26: 580-582.

2. Eliopoulos GM, Klimm K, Ferraro MJ, Jaeoby GA, Moellering RC: Comparative in vitro activity of piperaeillin combined with the beta-lactamase inhibitor tazobactam (YTR 830). Diagnostic Microbiology and Infectious Disease 1989, 12: 481--488. 3. Fass R J, Prior RB: Comparative in vitro activities of piperacillin-tazobactam and ticarcillin-clavulanate. Antimicrobial Agents and Chemotherapy 1989, 33: 1268-1274.

4. Fucks PC, Barry AL, Thornsberry C, Gavan TL, Jones RN: In vitro evaluation of Augmentin by broth microdilution and disk diffusion susceptibility testing: regression analysis, tentative interpretive criteria, and quality control limits. Antimicrobial Agents and Chemotherapy 1983, 24: 31-38. 5. Fucks PC, Barry AL, Thornsberry C, Jones RN: In vitro activity of ticarcillin plus clavulanic acid against 632 clinical isolates. Antimicrobial Agents and Chemotherapy 1984, 25: 392-394. 6. Gutman L, Kitzis MD, Yamabe S, Acar JF: Comparative evaluation of a new beta-lactamase inhibitor, YTR 830, combined with different beta-lactam antibiotics against bacteria harboring known beta-lactamases. Antimierobial Agents and Chemotherapy 1986, 29: 955--957.

7. Jacobs MR, Aronoff SC, Johenning S, Shlaes DM, Yamabe S: Comparative activities of the beta-taetamase inhibitors YTR 830, clavulanate, and sulbactam combined with ampicillin and broad spectrum penicillins against defined beta-laetamase-producing aerobic gram-negative bacilli. Antimicrobial Agents and Chemotherapy 1986, 29: 980-985.

8. Jones RN, Pfaller MA, Fuchs PC, Aldridge K, Allen SD, Gerlach EH: Piperacillin/tazobactam (YTR 830) combination: Comparative antimicrobial activity against 5889 recent aerobic clinical isolates and 60 Bacteroides fragilis group strains. Diagnostic Microbiology and Infectious Disease 1989, 12: 489--494.

9. Jones RN, Barry AL: Studies to optimize the in vitro testing of piperacillin combined with tazobactam (YTR 830). Diagnostic Microbiology and Infectious Disease 1989, 12: 495-510. 10. Kiastersky J, van der Auwerea P: In vitro activity of sulbactam in combination with various beta-lactam antibiotics. Diagnostic Microbiology and Infectious Disease 1989, 12, Supplement 4: 1655-1695. 11. Knapp CO, Sierra-Madero J, Washington JA: Activity of ticarcillin/clavulanate and piperacillin/tazobactam (YTR 830; CL-298, 741) against clinical isolates and against mutants derepressed for class I beta-lactamase. Diagnostic Microbiology and Infectious Disease 1989, 12: 511-515. 12. Mehter S, Drabu Y J, Blakemore PH: The in vitro activity of piperacillin/tazobactam, ciprofloxacin, ceftazidime and imipenem against multiple resistant gram-negative bacteria. Journal of Antimicrobial Chemotherapy 1990, 25: 915-919. 13. Sawai T, Yamaguehi A: Mechanism of beta-lactamase inhibition: differences between sulbactam and other inhibitors. Diagnostic Microbiology and Infectious Disease 1989, 12, Supplement 4: 1215-1295. 14. National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically M7-A2. NCCLS, Vil[anova, PA, 1990. 15. Sanders CC, laconis JP, Bodey GP, Samonis G: Resistance to ticarcillin-potassium clavulanate among clinical isolates of the family Enterobacteriaceae; role of PSE-1 beta-lactamase and high level of TEM-1 and SHV-1 and problems with false susceptibility in disk diffusion tests. Antimicrobial Agents and Chemotherapy 1988, 32: 1365-1369.

Effect of Subinhibitory Concentrations of Cefamandole and Cefuroxime on Adherence of Staphylococcus aureus and Staphylococcus epidermidis to Polystyrene Culture Plates H. Carsenti-Etesse*, J. D u r a n t , E. Bernard, V. Mondain, J. Entenza, P. D e l l a m o n i c a

T h e ability o f c e f a m a n d o l e and cefuroxime to inhibit adherence of staphylococci to polystyrene

culture plates was tested in an in vitro assay using eight strains each of Staphylococcus aureus and Staphylococcus epidermidis. The results indicated that subinhibitory concentrations o f cefamandole and cefuroxime altered the adDepartment of Infectious Diseases, Archet Hospital, BP 79, 06202 Nice Cedex 3, France.

Vol. 11, 1992

herence ability of both staphylococcal species, inhibition of adherence being more marked in the presence of cefamandole. It may be important to consider antiadherence properties in association with bactericidal activity when selecting agents for antibiotic prophylaxis.

Infection associated with the use of implanted biomaterials can constitute a serious complication. Colonization of a biomedical implant can be divided into several phases consisting of adherence, growth and slime production (1). Bacterial adherence appears to be controlled in vitro by hydrophobic and electrostatic surface interactions between bacteria and the substrate (2). Host tissue or plasma proteins deposited on the surfaces of medical implants may be important determinants of bacterial adhesion in vivo. The matrix of bacteria, slime and exogenous factors (biofilm layer) which envelops the surface of a colonized implant firmly anchors adherent bacteria to the surface of the device (3, 4), and protects adherent bacteria from host defences (5). Antimicrobial therapy alone, even with agents demonstrating activity in vitro against the offending strain, is seldom an effective means of sterilizing the surface of an infected implant (6) and removal of infected devices is often required. Strains most often recovered from infected implants are Staphylococcus aureus and Staphylococcus epidermidis (4). Previous studies have shown that subinhibitory concentrations of certain antimicrobial agents influence (both positively and negatively) the adherence of a variety of microorganisms (7, 8). In view of the potential quality of cefamandole and cefuroxime in the prophylaxis of biomaterial implant infections, we examined the influence of subinhibitory concentrations of each agent on the adherence of clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis to polystyrene culture plates.

Materials and Methods. Eight clinical strains of Staphylococcus aureus and Staphylococcus epiderrnidis respectively were selected for the study due to their adherence properties. All Staphylococcus epidermidis were identified by the API Staph Ident System (Analytab Products, USA). To avoid loss of stability in slime and adherence tests by serial transfers on media, the original strain in suspension was divided into aliquots kept at -70 *C and one aliquot was used for each experiment. Cefamandole was obtained


from Eli Lilly, France and cefuroxime from Glaxo, UK. MICs for the bacterial isolates were determined by a microdilution method under the same conditions with which adherence measurements were made. Susceptibility tests were performed in Mueller Hinton (MI-I) broth with inocula of 106 cfu/ml containing twofold serial dilutions of antibiotic solutions from 128 to 0.12 mg/l. The MIC was defined as the lowest concentration of antibiotic which inhibited visible growth after overnight incubation at 37 °C. The ability of strains to produce slime was tested by the qualitative tube assay described by Davenport et al. (9). Overnight, growth in broth was decanted and the glass tubes were allowed to dry. The internal walls of each tube were washed with Gram-safranin solution, and the amount of stained slime adhering to the walls was semiquantitated as 0 (absent), +, ++ or +++ compared to positive and negative controls. Adherence measurements were performed by a spectrophotometer method in 96-well microtiter plates (Nunc, Denmark) (10). Adherence tests were performed without antibiotic in MH and TS (Trypticase soy) broths. Tests with antibiotics were performed in M H broth. Overnight cultures were adjusted to 106 cfu/ml and diluted with serial twofold dilutions of drugs so that final concentrations were equal to 112 to 1/16 the MIC for the strain. Cultures without antibiotic served as negative controls. Following incubation for 6 h at 37 °C, bacterial growth was decanted from the plate. Each well was washed four times with phosphate buffer o f p H 7 to remove non-adherent bacteria. The remaining attached bacteria were fixed in absolute ethanol, dried and stained with crystal violet (Merck, Germany). Excess stain was removed with running tap water. The absorbance was measured at 570 nm using a micro-ELISA Auto Reader (Dynatech Laboratories, USA). Each experiment was run in quadruplicate and repeated at least three times. A mean adherence value was then calculated for each concentration and compared to the value of the control using the two-tailed Student's t-test.

Results and Discussion. The results showed a significant increase of bacterial adherence for only four of eight Staphylococcus aureus strains in TS broth (2 positive and 2 negative slime producers). However, mean optical density values did not show any significant difference when tests were performed in M H or TS broth after 6 or 24 hours incubation. For Staphylococcus epidermidis the optical density value after 6 hours of incubation


Eur. J. Clin. Microbiol. Infect. Dis.

was significantly increased in TS broth for only one of the six strains tested. M e a n optical density values were not significantly different after incubation for 6 hours in M H or TS broth. A f t e r incubation for 24 hours optical density values were considerably increased in both M H and TS broth (Table 1). A t 6 hours, slime production enhanced by glucose content of TS b r o t h did not seem to interfere with adherence properties, since some strains which were high producers of slime did not show any significant increase in adherence. As the focus of our study was on adherence properties, the choice of a m e d i u m without glucose such as M H broth was appropriate to avoid rapid slime production which results in marked standard deviations in optical density values at 24 hours. Table 2 shows the M I C values related to slime production for the strains tested. All strains were susceptible to 2/ag/ml or less of cefamandole and to 16 ~tg/ml or less of cefuroxime. A d h e r e n c e results were expressed as the mean percentage adherence values of treated cultures c o m p a r e d with non-treated controls (Table 2), For Staphylococcus aureus at one fourth the M I C

cefamandole was active against seven of eight strains but cefuroxime showed loss of activity, inhibiting adherence by m o r e than 20 % in only two of eight strains. A t one-eighth the M I C cefuroxime showed no activity whereas cefamandole maintained activity against three of eight Staphylococcus epiderrnidis, strains. For cefuroxime and cefamandole inhibited adherence of all the strains tested at o n e - f o u r t h the MIC. H o w e v e r , at one-eighth the M I C only cefamandole was still active against all strains; cefuroxime inhibited adherence by m o r e than 20 % in five of eight strains. In kinetic studies Christensen et al. (1) showed that colonization of s m o o t h surfaces proceeded through an initial adherence phase, which was followed by an accumulation phase, and that the antigen associated with slime production was only expressed by slime producing strains. Peters et al. (4) showed that slime production is not necessarily involved in the initial phase of attachment. Younger et al. (11) showed that adherence measurements may vary, depending on the glucose content of the TS broth, a few organisms

Table 1: Adh~r~nc~va~u~s(opticaldcnsity)~btaincdaftcr6and24h~urs~fincubati~n~f~ightstrainscach~fStaPhy~c~c-

cus aureus and Staphyloccus epidermidis inMueller-Hinton (MH) and Tryp ticase soy (TS) broth. Results are expressed as the mean with standard deviation. Strain

Slime production

6 hours MHbroth

S. aureus 3 47 71 80 96 183 192 195 202 203

+++ + ++ 0 0 ++ + 0

0 +

4.9 9.0 19.1 17,4 33.2 27.2 36.1 11.3 11.7 13.1

143.2 ± 30.9


S. epidermidis 2 16 77 93 104 127

180.8 ± 134.6 ± 193.7 ± 147.4 ± 156.3 ± 128.7 ± 146.2 ± 122.8 ± 88.7 ± 126.6 ±

0 +++ +++ + 0 ++

Mean a Significant difference. bCluster formation.

202.8 ± 158.0 ± 171.9 ± 164.7 ± 153.0 ± 108.7 ±

3.4 19.4 31.9 14.7 12.2 12.5

159.8 ± 30.6

24 hours TSbroth 242.6 ± 109.5 ± 211.4 ± 204.7 ± 191.2 ± 320.0 ± 148.2 ± 98.6 ± 94.7 ± 130.2 ±

7.6a 17.4a 30.6 26.4a 19.0 179.4 18 5.1a 2.6 8.8

MHbroth 104.9 ± 170.0 ± 190.8 ± 235.7 ± 210.2 ± 92.5 ± 117.1 ± 222.1 ± 146.0 ± 169.9 ±

26.2 18.9 22.5 22.5 105.6 5.9 39.2 100.8 41.6 22.9

174.6 ± 72.8

165.9 ± 50.0

173.1 ± 26.4 163.8 ± 118.9 240.7 ± 44.3 198.5 -+ 13.6 125.1 + 13.0 181.9 ± 20.7a

462.6 257.6 ± 806.5 ± 200.9 ± 367.9 ± 422.6 ±

180.5 ± 38.3

418.9 ± 213.9

34.2 188.6 30.0 11.6 111.1

TSbroth 119.6 ± 320.5 ± 179.6 ± 274.1 ± 277.0 ± 181.0 ± 148.9 ± 222.3 ± 147.6 ± 229.4 ±

76.4 72.6 23.0 28.0 50.5 26.7a 13.0 66.1 39.4 48.8

210.0 ± 65.7 879.5 445.7 ± 95.6 ± 158.7 ± 1200.0 1111.3 ±

55.9 5.0b 37.5 156.8

759.1 ± 444.0

V o l . 11, 1992


Table 2: Mean percentage adherence values with standard deviation for eight Staphylococcus aureus and Staphylococcus epidermidis strains exposed to subinhibitory concentrations of cefamandole and cefuroxime. Strain

Slime production

Cefamandole MIC ~

S. aureus 3 47 71 80 96 159 183 192

+++ + ++ 0 0 0 ++ +

1 2 2 2 0.5 2 1 2


S. epidermidis 2 16 70 77 93 104 127 131


0 +++ ++ +++ + 0 ++ +++

1 2 0.25 2 0.25 0.5 0.25 2


1/4 MIC 53.9 101.7 48.8 40.8 52.2 43.7 75.6 11.3

± ± ± ± ± ± ± ±

1/8 MIC

19.5b 91.6 ± 11.3 26.8 113.8 ± 26.7 9.8b 73.6 ± 14.0b 9.9b 62.0 ± 13.6b 10.7 b 82.9 ± 16.0 12.2b 91.4 ± 13.8 12.2 b 86.4 ± 12.0 4.3b 53.1 ± 29.4 b

53.5 ± 26.4 b

81.9 ± 18.9

50.5 41.4 26.0 12.7 52.9 49.0 42.9 75.5

68.1 51.7 61.3 42.3 62.6 63.5 59.8 77.4

± ± ± ± ± ± ± ±

11.0b 10.3b 5.5 b 2,6b 6.4b 10.9b 5.2b 15.2b

46.4 ± 18.6 u

± ± ± ± ± ± ± ±

14.6 b 13.4b 10.5b 10.1b 8.4 b 15.0 11.8 b 6.9 b

60.8 ± 11.3 b

MIC a 2 2 2 2 2 2 2 2

1/4 MIC 75.1 95.1 74.5 103.6 109.0 94.3 80.5 88.0

± 12.8 b

± ± ± ± + ± ±

11.4 13.8 b 16.0 12.3 11.6 10.8 b 8.2

90.0 ± 12,8 1 0.5 2 16 0.25 0.25 0.25 1

57.4 63.8 16.2 7.3 63.4 68.1 59.1 78.4

± ± ± ± ± ± ±

12.1 b 14.1 b 5.0 b 2.2 b 7.6 b 19.0 b 6.8 b

± 11.0 b

51.7 ± 25.6 b

1/8 MIC 110.5 124.7 83.5 104.4 108.3 93.4 92.6 101.5

± + ± ± ± ± ± ±

15,2 24.5 14.5 18,3 8.7 14.9 9.7 15.0

102.4 ± 12.7 69.8 85.5 63.6 7.4 84.3 78.7 73.9 92.0

± ± ± ± ± ± ± ±

13.8b 10.0 9.6b 2,6 b 8.5 b 10.8 b 13.7 b 8.1

69.4 ± 26.7 b

a MIC values expressed in mg/ml. bp < 0.05 (significant difference versus untreated cultures).

b e i n g m o r e a d h e r e n t in n o n - g l u c o s e c o n t a i n i n g m e d i a . S h a d o w e t al. (12) r e p o r t e d g r e a t e s t a d herence for one strain regardless of whether g l u c o s e w a s p r e s e n t in t h e c u l t u r e b r o t h . C h r i s t e n s e n e t al. (13) r e p o r t e d t h a t in b r o t h to encourage slime production, the microorganisms c o a g g l u t i n a t e i n t o a m a s s . T h i s p h e n o m e n o n was o b s e r v e d in o u r s t u d y in o n e s l i m e - p r o d u c i n g Staphylococcus epidermidis s t r a i n w i t h v i s i b l e c l u s t e r s at 24 h o u r s a n d a d e c r e a s e in a d h e r e n c e p r o p e r t i e s in T S b r o t h r e l a t e d to t h e c o a g g l u t i n a t i o n ( T a b l e 1). O u r e x p e r i m e n t s w e r e p e r f o r m e d a f t e r 6 h o u r s o f i n c u b a t i o n to a v o i d s u c h c o a g glutination. I n o u r s t u d y e a c h s t r a i n was a f f e c t e d d i f f e r e n t l y by the drugs and inhibition of adherence did not correlate with the property of slime production. T h i s o b s e r v a t i o n is in a g r e e m e n t w i t h e a r l i e r findings (14). H a a g e n et al. (15) r e p o r t e d t h a t s l i m e p r o d u c t i o n d i d n o t s e e m to b e i n v o l v e d in a d h e r e n c e b e c a u s e Staphylococcus" aureus i s o l a t e s did not produce slime but nevertheless adhered to v a r i o u s s u r f a c e s . T h u s t h e p r o d u c t i o n o f a s l i m y m a t r i x in w h i c h b a c t e r i a b e c o m e e m b e d d e d m a y e s s e n t i a l l y b e a p r o c e s s to a c h i e v e m o r e

stable attachment once adherence has taken p l a c e . S l i m e p r o d u c t i o n b y Staphylococcus epidermidis is v i e w e d as a v i r u l e n c e f a c t o r promoting infections associated with prosthetic d e v i c e s (16). T h e i n c o n s i s t e n t s u c c e s s of a n t i m i c r o b i a l t h e r a p y of prosthetic implant infection may reflect poor penetration of antibiotics through a biofilm m a t r i x a n d e x p o s u r e o f a d h e r e n t o r g a n i s m s to s u b i n h i b i t o r y c o n c e n t r a t i o n s o f p o t e n t i a l l y effective a g e n t s . D u n n e (17) s h o w e d t h a t at s u b i n hibitory concentrations vancomycin and c e f a m a n d o l e c o u l d p o t e n t i a l l y s t i m u l a t e t h e formation of a biofilm by coagulase-negative s t a p h y l o c o c c i a t t a c h e d to h y d r o p h o b i c surfaces. E v a n s et al. (6) s h o w e d t h a t v a n c o m y c i n f a i l e d to kill t h e b i o f i l m o r g a n i s m s a s s o c i a t e d w i t h an indwelling peritoneal catheter. In view of the serious consequences of prosthetic i n f e c t i o n s , p r e v e n t i o n of s u c h i n f e c t i o n s is o f p r i m e i m p o r t a n c e . Staphylococcus aureus a n d Staphylococcus epidermidis a r e t h e p a t h o g e n s m o s t f r e q u e n t l y e n c o u n t e r e d in c a t h e t e r ass o c i a t e d i n f e c t i o n s . B a c t e r i a l a d h e r e n c e to artificial s u r f a c e s m a y b e a c r u c i a l e a r l y s t e p in in-


travenous device infections. Certain antimicrobial agents might suppress expression of the bacterial surface adhesin at doses m u c h lower than those n e e d e d to kill organisms or stop their growth (18). Sublethal concentrations of antibiotics m a y exert their antiadherence effects by p e r t u r b a t i o n o f the bacterial surface structures (14). Lorian (7) r e p o r t e d that exposure of staphylococci to b e t a - l a c t a m s and lincomycin can p r o d u c e a b n o r m a l , larger forms. It has b e e n r e p o r t e d that morphologic changes in bacteria ceils and structural changes of peptidoglycan occur in staphylococci grown in m e d i a containing low concentrations of b e t a - l a c t a m antibiotic, rendering organisms m o r e susceptible to phagocytosis (19). Only a limited n u m b e r of studies have dealt with the effects of subinhibitory concentrations of antimicrobial agents on the a d h e r e n c e of staphylococci. Shadow et al. (12) showed that cell-wall acting agents, particularly polymyxin B and b e t a - l a c t a m antibiotics, cephalothin and imipenem, p r o d u c e d the greatest inhibition of adh e r e n c e of t h r e e strains of coagulase negative staphylococci on tissue culture plates. Beta-lact a m antibiotics such as c e f a m a n d o l e and cefuroxime are often used as antistaphylococcal agents in antibiotic prophylaxis (20). T h e y m a y play an important role in preventing infection by two mechanisms: inhibition of adherence and a u g m e n t a t i o n of host defense by rendering organisms m o r e susceptible to intracellular killing (19). T h e inoculum a m o u n t responsible for infection during surgery is p r o b a b l y low. ¥ourassowsky et al. (21) showed that c e f a m a n d o l e was m o r e active t h a n c e f u r o x i m e against Staphylococcus aureus and Staphylococcus epidermidis under these conditions. These properties in association with e n h a n c e d bactericidal activity (22) and inhibition of adherence should b e considered when choosing a cephalosporin for antibiotic prophylaxis. References 1. Christensen GD, Barker LP, Mawhinney TP, Baddour LM, Simpson WA: Identification of an antigenic marker of slime production for Staphylococcus epidermidis. Infection and Immunity 1990, 58: 2906-2911. 2. Chri~ensen GD, Simpson WA, Bisno AL, Beachey EH: Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces. Infection and Immunity 1982, 37: 318-326. 3. Franson TR, Sheth NK, Rose HD, Sohnle PG: Scanning electron microscopy of bacteria adherent to intravascular catheters. Journal of Clinical Microbiology 1984, 20: 500-505.

Eur. J. Clin. Microbiol. Infect. Dis.

4. Peters G, Locci R, Pulverer G: Adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters. Journal of Infectious Diseases 1982, 146: 479-482. 5. Johnson GM, Lee DA, Regehnann WE, Gray ED, Peters G, Quie PG: Interference with granulocyte function by Staphylococcus epidermidis slime. Infection and Immunity 1986, 54: 13-20, 6. Evans RC, Holmes C.J: Effect of vancomycin hydrochloride on Staphylococcus epidermidis biofilm associated with silicone elastomer. Antimicrobial Agents and Chemotherapy 1987, 31: 889-894. 7. Lorian V: Effect of low antibiotic concentrations on bacteria: effects on ultrastructure, their virulence, and susceptibility to immune defenses. In: Lorian, V (ed): Antibiotics in laboratory medicine. Williams & Wilkins, Baltimore, 1986, p. 596-668, 8. Ofek I, Beachey EH, Eisenstein BI, Aikan ML, Sharon N: Suppression of bacterial adherence by subminimal inhibitory concentrations of 15-1aetam and aminoglycoside antibiotics. Reviews of Infectious Diseases, 1979, 1" 832-837. 9. Davenport DS, Massanari RM, Pfaller MA, Bale MJ, Streed SA, Hierholzer WJ: Usefulness of a test for slime production as a marker for clinically significant infections with coagulase-negative staphylococci. Journal of Infectious Diseases 1986, 153: 332-339. 10. Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, Beachey EH: Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. Journal of Clinical Microbiology 1985, 22: 996-1006. 11. Younger JJ, Christensen GD, Bartley DL, Simmons JCH, Barrett FF: Coagulase-negative staphylococci isolated from cerebrospinal fluid shunts: importance of slime production, species identification, and shunt removal to clinical outcome. Journal of Infections Diseases 1987, 156: 548-554. 12. Schadow K, Simpson WA, Chrislensen GD: Characteristics of the adherence to plastic tissue culture plates of coagulase-negative staphylococci exposed to subinhibitory concentrations of antimicrobial agents. Journal of Infectious Diseases 1988, 157: 71-77. 13. Christensen GD, Parisi JT, Bisno AL, Simpson WA, Beachey EH: Characterization of clinically significant strains of coagulase-negative staphylococci. Journal of Clinical Microbiology 1983, 18: 258-269. 14. Shibl AM: Influence of subinhibitory concentrations of antibiotics on virulence of staphylococci. Reviews of Infectious Diseases 1987, 9: 704-712. 15. Haagen IA, Heezius HC, Verkooyen RP, Verhoef J, Verbrugh HA: Adherence of peritonitis-causing staphylococci to human peritoneal mesothelial cell monolayers. Journal of Infectious Diseases 1990, 161: 266-273. 16. Etesse-Carsenti H, Entenza J, Bernard E, Dellamonica P: Propri6t6s d'adh6rence et production de slime des staphylocoques: relation avecla pathog6nicit6. Pathologic Biologic 1990, 36: 249-254. 17. Dunne WM: Effects of subinhibitory concentrations of vancomycin or cefamandole on biofilm production by coagulase-negative staphylococci. Antimicrobial Agents and Chemotherapy 1990, 34: 390--393. 18. Schifferli DM, Beachey EH: Bacterial adhesion: modulation by antibiotics with primary targets other than protein synthesis. Antimierobial Agents and Chemotherapy 1988, 32, 11: 1609-1613.

Vol. 11, 1992

19. Milatovie D, Braveny I, Verhoef J: Clindamycin enhances opsonization of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 1983, 24: 413417. 20. SlamaTG, Sklar S,I,MisinskiJ, Fess SW: Randomized comparison of cefamandole, cefazolin and cefuroxime prophylaxis in open-heart surgery. Antimicrobial Agents and Chemotherapy 1986, 29: 744-747. 21. Yourassowsky E, Van Der Linden MP, Crokaert F: Inoculum effect on growth-delay time of oxaeillinresistant strains of Staphylococcus aureus and Staphylococcus epidermidis exposed to cefamandole, cefazolin and cefuroxime. Antimicrobial Agents and Chemotherapy 1990, 34: 505-509. 22. Stratton CW, Liu C, Weeks IS: Activity of LY 146032 compared with that of methieillin, cefazolin, cefamandole, cefuroxime, ciprofloxacin and vancomycin against staphylococci as determined by killing-kinetic studies. Antimicrobial Agents and Chemotherapy 1987, 31: 1210-1215.

Evaluation of a Monoclonal Antibody for Detection of Helicobacterpylori in a Direct Immunofluorescence Test U. R o d e w i g 1, W. B e m b 4, D. Bitter-Suermann 1, M. Elsheikh 1, S. Fritsch 2, E. G l e n n - C a l v o 1, B. S o u d a h 2, M. Varrentrapp 3, S. Wagner 3, W. B~tr 1.

A monoclonal antibody was developed for detection of Helicobacter pylori in gastric tissue sections in a direct immunofluorescence test. On a comparison of the immunofluorescence test with standard methods for detection of HeIicobacter pylori, i.e. culture, the urease activity test and histological examination of tissue sections, using 158 biopsy specimens, 30 specimens were positive in all methods and 64 negative. In the remaining cases comparison was not possible because either immunofluorescence (29 specimens) or the standard methods (16 specimens) gave ambiguous results. The direct immunofluorescence test may have potential as an alternative to standard methods, but further testing in a defined patient population is necessary.

1Institute of Medical Microbiology,2Institute of Pathology and 3Department of Gastroenterology, Medical School Hannover, 3000 Hannover 61, Germany. 4Institute of Medical Microbiology,University Clinic, 6500 Mainz, Germany.


The main procedures currently used for detection of Helicobacterpylori are culture, histological examination and detection of urease activity (1). However, a disadvantage-of these techniques is that they have low sensitivity (2) so that a correct diagnosis may not be established in a number of cases. Moreover, these techniques are cumbersome to use. There is thus a need for a method for detection of Helicobacter pylori that is both convenient to use, sensitive and specific. Direct immunofluorescence using monoclonal antibodies (MABs) might seem to meet these demands. Until now there have been few reports on the production of MABs against Helicobacter pylori or the use of MABs for its identification. Indirect immunofluorescence tests (3, 4) and an immunoperoxidase method have been introduced which use MABs (5), however in all cases the epitopes are formaldehyde-sensitive so that the handling of biopsy material in these techniques has proved complicated. We therefore developed an M A B directed against Helicobacter pylori lipopolysaccharide and tested it in a direct immunofluorescence method (DIF) using gastric biopsy specimens.

Materials and Methods. Helicobacter pylori was isolated from gastric biopsies and identified using standard methods (1, 2). Monoclonal antibody was produced as described elsewhere (6). Balb/c mice were immunized i.p. with a suspension of three clinical isolates of viable Helicobacterpylori (4 x 107 bacteria per strain). Antibody titers were determined with an enzyme immunoassay (EIA) using whole bacteria (6 x 106 bacteria/well) as antigen. To determine specificity of the MAB, Helicobacter pylori and a panel of other bacteria including other Helicobacter species, Campylobacter and Wolinella species was used. The E I A was performed as described elsewhere (6). Bacterial protein was analyzed by SDS-PAGE (7) using 1.2 x 108 cells/lane. To demonstrate Helicobacter pylori lipopolysaccharide (LPS), phenol extracts were separated on SDS-PAGE using a 15 % slab gel with 4M urea (8). The gels were stained with silver (9). In a Western blot the gels were transferred electrophoretically to nitrocellulose sheets by standard methods (6) and developed with peroxidase-labelled goat anti-mouse serum (Dianova, Germany) using orthonaphthol as substrate with 3 % 1-1202. In the direct immunofluorescence (DIF) method the MAB, designated MAB 2107, was coupled

Effect of subinhibitory concentrations of cefamandole and cefuroxime on adherence of Staphylococcus aureus and Staphylococcus epidermidis to polystyrene culture plates.

The ability of cefamandole and cefuroxime to inhibit adherence of staphylococci to polystyrene culture plates was tested in an in vitro assay using ei...
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