Journal of Chemotherapy

ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20

In Vitro Activity of Cefpirome (HR 810) against Enterococci and Staphylococci E. Piacentini, G. Amalfitano, M. Ligozzi, P. Canepari & R. Fontana To cite this article: E. Piacentini, G. Amalfitano, M. Ligozzi, P. Canepari & R. Fontana (1992) In Vitro Activity of Cefpirome (HR 810) against Enterococci and Staphylococci, Journal of Chemotherapy, 4:6, 338-341, DOI: 10.1080/1120009X.1992.11739188 To link to this article: https://doi.org/10.1080/1120009X.1992.11739188

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Journal of Chemotherapy

In Vitro Activity of Cefpirome (HR 810) against Enterococci and Staphylococci E. PIACENTINI - G . AMALFITANO M. LIGOZZI- P. CANEPARI- R. FONTANA

Summary ------------------------ --------The inhibitory activity of cefpirome (HR 810), a new cephalosporin derivative for parenteral use, was tested by agar dilution methods against Enterococcus fsecalis (100 strains), Stsphylococcus sureus (40 strains) and coagulase-negative staphylococcal species (60 strains) in comparison with other betalactam antibiotics. For E. fsecslis, the cefpirome minimum inhibitory concentration (MIC) range was 2-128 Jlg/ml, with an MIC,. of 8 Jll/ml, and an MIC, 0 of 64 Jlg/ml. The optimal bactericidal activity against strains with MICs of :::s 8 Jlg/ml occurred at 2· 4 times the MIC, and the reduction in the initial inoculum was 99.9-99.7% after 24 h incubation at these concentrations. Mec gene-negative staphylococci (both S. sureus and coagulase- negative species) had cefpirome MICs of 0.2.5-2 Jlg/ml (MIC ,. 0 ..5 Jlg/ml, MIC,. lJlg/ml). Mec gene-positive strains had MICs of 0 ..5-128 Jlg/ml (MIC,. 2 Jlg/ml, MIC,. 32 Jlg/ml). Strains with borderline resistance to oxacillin which did not harbor the mec gene and which were susceptible to cefpirome maintained their susceptibility even when highdensity inocula were used and after several passages in media containing the antibiotic.

Institute of Microbiology, University of Verona, Verona, Italy. CotTespondence: Prof. Roberta Fontana, Universita di Verona, Istituto di Microbiologia, Strada Le Grazie, 37134 Verona, Italy. © Eclizioni Riviste Scientifiche . Firenze

Vol. 4 - n. 6 (338-341) - 1992

These studies present some potential advantages of cefpirome over other cephalosporin& in the inhibitory activity against Gram-positive cocci. Key words: cefpirome, enterococci, staphylococci.

INTRODUCTION

Nosocomial infections due to enterococci and staphylococci are on the increase and represent a serious therapeutic problem as most of these organisms are resistant to several antibiotics, particularly beta-lactams 1 • 2 • Diminished susceptibility of Gram-positive bacteria to beta-lactams is essentially due to production of penicillinases (seldom in enterococci, often in staphylococci) and/or synthesis of penicillin binding proteins (PBPs) with a low affinity for these antibiotics (both enterococci and staphylococci) 3-6 . Resistance due to penicillinases can be overcome by several beta· lactams which are resistant to the hydrolytic activity of these enzymes, but beta-lactams are practically inactive against strains producing large amounts of low-affinity PBPs. Development of new beta-lactams with an improved spectrum of activity including intrinsically resistant Gram-positive cocci is a major goal in pharmaceutical research. In this study, we investigated the anti-enterococcal and antistaphylococcal activity of cefpirome (HR 810), an amino-2-thiazolyl cephalosporin with a methyl-cyclopentapyridinium hydroxide substituted in position 3 of cephem nucleus, which should improve the spectrum of activity relative to other aminothiazolyl cephalosporins. The spectrum of this very recent generation cephalosporin has been shown to include ISSN 1120-009X

339

IN VITRO ACTIVITY OF CEFPIROME (HR 810) AGAINST ENTEROCOCCI AND STAPHYLOCOCCI

members of the family Enterobacteriaceae,

Pseudomonas aeruginosae, Staphylococcus aureus and Enterococci 7 • MATERIALS AND METHODS

Strains. The organisms tested were clinical isolates collected at the Microbiology Laboratories of the Institute of Microbiology of Verona University and Ospedale Maggiore. Identification was performed by means of the API Strep and API Staph micromethods (Analytical Profile Index System S.A.). Strains were stored in sterile glycerol at -20° C. All the staphylococcal isolates were examined for the presence of the gene (mec) responsible for intrinsic resistance to beta-lactams, using a polymerase chain reaction (PCR) -based protocol 8 • The 200 isolates were distributed as follows: 100 Enterococcus faecalis, 46 Staphylococcus aureus, 48 Staphylococcus epidermidis, 4 Staphylococcus hominis, 2 Staphylococcus xylosus, 2 Staphylococcus capitis, and 1 Staphylococcus cohnii.

Antimicrobial agents. Cefpirome (HR 810) was supplied by Roussel Pharma (Milan, Italy), cefotaxime by Hoechst (Scoppito, Italy), cefuroxime and ceftazidime by Glaxo (Verona, Italy), ceftriaxone by Hoffmann-La Roche (Basel, CH), ampicillin by Squibb (Anagni, Italy), oxacillin by Sigma (St. Louis, USA), and vancomycin by Lilly (Sesto Fiorentino, Italy) . Benzyl- 14 C- penicillin (59 mCi/mmol) was purchased from the Amersham Radiochemical Centre (UK) .

Bacteria were grown in MHB containing antibiotic concentrations ranging from the MIC to 32 x MIC, and colonies were counted after 24 h incubation at 37° C.

Penicillin binding protein analysis. The affinity of cefpirome for PBPs was measured as previously described 6 •

RESULTS

The antimicrobial activity of cefpirome against 100 E. faecalis isolates is compared with that of the other beta-lactams and vancomycin in Table 1. The new drug was less active than ampicillin and vancomycin but was the most active cephalosporin tested. The mean MIC,o was 8 !lg/rnl, i.e. 8-16-fold lower than with ceftazidime and ceftriaxone. Cefpirome showed good bactericidal activity against strains with such MICs, the optimal bactericidal concentration being 2-4 x MIC . At these concentrations the reduction in the initial inoculum ranged from 99.9 to 99.7% after 24 h incubation. The bactericidal activity of cefpirome decreased at higher antibiotic concentrations (paradoxical effect), as is often the case of penicillins against enterococci 9 • To understand the mechanism of the antienterococcal activity, the affinity of cefpirome for PBPs of enterococci was investigated . Table 2 shows that, like the other cephalosporins tested, cefpirome had a low affinity for PBPs 4, 5 and 6 of E. hirae (faecium) ATCC 9790, a strain used in several studies regarding the mechanism of action of beta-lactams on members of this

Susceptibility testing. MICs were determined by the agar dilution method. The medium was Mueller Hinton agar (MHA, Difco Laboratories) supplemented with 2% NaCI for the evaluation of staphylococcal susceptibility. The inoculum was 10 4 CFU/spot and was applied to the surface with the aid of a Steers replica tor. Incubation was performed for 24 hat 37° C. The MIC was read as the lowest antibiotic concentration completely inhibiting bacterial growth. MBC was determined as described elsewhere 9 •

TABLE 1 - Activity of cefpirome against E. faecalis (n = 100) . MIC (!!g/ml) Antibiotic

Cefpirome Ceftazidime Ceftriaxone Ampicillin Vancomycin

range

50%

2-128 128-> 128 8-> 128 0.5 -8 0.5-2

8 128

64 2 1

90%

64 > 128 > 128 8 2

Susceptibility was determined by the agar dilution method in Mueller-Hinton agar using an inoculum of 10' CFU/spot. MICs were read after 24 h incubation at 37° C .

340

E. PIACENTINI - G. AMALFITANO - M. LIGOZZI - P. CANEPARI - R. FONTANA

TABLE 2 - Interaction of cefpirome and other beta-lactams with PBPs of E. hirae (faecium) ATCC 9790.

Minimal concentration (J.lg/ml) saturating PBP Antibiotic

Cefpirome Ceftazidime Ceftriaxone Ampicillin

TABLE

4 32 32 0.8

2

3

4 16 16 0.8

2 8 8 0.8

4

5

6

>256 >256 >256 >256 >256 >256 >256 >256 >256 32 16 1.6

3 -Activity of cefpirome against staphylococci (n = 100). MIC (J.lg/ml)

Organisms

Mec-negative staphylococci (n=54)

Mec-positive staphylococci (n=44)

Antibiotic range

50%

90%

Cefpirome Cefotaxime Cefuroxime Oxacillin Vancomycin

::50.25-2 ::50.25-8 ::50.25-8 0.25-8 1-4

0.5 0.5 0.25 0.5 2

1 2

Cefpirome Cefotaxime Cefuroxime Oxacillin Vancomycin

0.5-> 128 0.5-> 128 0.5-> 128 4-> 128 1-4

2 4 2 32 2

32 64 128 > 128 4

4 4

Susceptibility was determined by the agar dilu9on method in Mueller- Hinton agar containing 2% NaCl, using an inoculum of 10' CFU/spot. MICs were read after 24 h incubation at 32° c.

TABLE

genus 5 • 6 • In contrast, cefpirome showed higher affinity than the other cephalosporins for PBPs 1, 2 and 3, which are considered essential for cell growth and division 10 • The MIC range, MICso and MIC9o of cefpirome against the staphylococcal strains both with and without the mec gene are shown in Table 3. The new drug was very active against the mec- negative strains and also showed a good activity against 50% of the mec-positive strains (MICso = 2 l!g/ml). Five strains which were susceptible to cefpirome but resistant or borderline-resistant to oxacillin were further analyzed with regard to betalactamase activity 11 , the inoculum effect on susceptibility and mutational frequency in terms of cefpirome resistance. The three strains which harbored the mec gene exhibited a substantial increase in MIC when high-density inocula were used in susceptibility tests. The populations initially contained a small fraction of resistant cells (8 l!g/ml), and mutants with a higher level of resistance (64 l!g/ml) were occasionally isolated after only a few passages at increasing antibiotic concentrations. In contrast, two borderline strains which did not. harbor the mec gene but did produce beta-lactamase remained susceptible to cefpirome even when high-density inocula were used, and no resistant mutants were isolated after several passages in media containing the antibiotic (Table 4). It should be noted that S. epidermidis 38, a non-producer of beta-lactamase, had the same MIC for oxacillin and cefpirome.

4 - Properties of oxacillin-resistant and borderline-resistant staphylococcal strains susceptible to cefpirome.

Meca gene

Strain

Betalactamaseb

O xacillin MICe ().lg/ml)

10' 10' CFU/spot CFU/plate S. S. S. S. S. S.

epidermidis epidermidis aureus epidermidis epidermidis epidermidis

8 35 73

5 24 38

+ + +

+ + + + +

16 16 32 8 8 0.25

Cefpirome MICe (J.lg/ml)/ resistant mutant frequency after no. passage

Cefpirome MICe (J.lg/ml) with inoculum of

2 2 2 1 0.25

a identified using a PCR-based protocol b beta-lactamase activity was determined by the nitrocefin method c MICs were determined as described in Table 3.

8 16 16 2 2 0.25

1

2

•3

64/10'' 128/10' 1 128/10' 7 2 2 0.25

128/10' 1 nd nd 2 2 0.25

nd nd nd 8/10 ' ' 8/10' ' 0.25

IN VITRO

ACTIVITY OF CEFPIROME (HR 810) AGAINST ENTEROCOCCI AND STAPHYLOCOCCI DISCUSSION

The good activity of cefpirome against enterococci (MICso 8 J..tg/ml) relative to the other cephalosporins tested (MICso 64 J..tg/ml) is an interesting feature of this drug. Such activity may prevent superinfections due to enterococci, which can occur during therapy with the latest generation of cephalosporins 12 • In addition, resistance to the hydrolytic activity of beta-lactamases should make cefpirome active against enterococcal strains producing this enzyme. The bactericidal activity of cefpirome against susceptible populations (MIC s 8 ~-tg/ml) was optimal at 2-4 x MIC and was very close to the 99.9% decrease in the initial inoculum taken to define bactericidal activity 13 • In this respect, cefpirome behaved like ampicillin and other beta-lactams active against enterococci. Cefpirome was very active against oxacillinsusceptible staphylococci, and against at least 50% of the oxacillin-resistant strains. However, a distinction should be made among oxacillin-resistant cefpirome- susceptible strains on the basis of the presence of the mec gene. Indeed, it was found that mec gene-positive strains were resistant to cefpirome when highdensity inocula were used for the test and that mutants with a high level of resistance were isolated after only a few passages in media containing the drug. In contrast, mec gene-negative strains remained susceptible to cefpirome even when high-density inocula were used in the assay, and few resistant mutants were isolated after several passages in media containing subinhibitory concentrations of the drug. These results partly support the general view that cephalosporins should not be used for the treatment of infections caused by oxacillin-resistant strains despite their activity in vitro. In addition, they stress the need for correct identification of the resistance genotype or phenotype before excluding potentially useful, non-toxic drugs such as beta-lactams from the management of staphylococcal infections. In conclusion, we consider cefpirome a very promising parenteral cephalosporin, which pro-

341

vides more potent activity than third generation cephalosporins against Gram-positive species. ACKNOWLEDGEMENTS - This work was supported by Consiglio Nazionale delle Ricerche, Progetto Finalizzato Fattori di Malattia, grant No. 92.00038.PF41.

REFERENCES 1 Chambers HF. Methicillin-resistant Staphylococci. Clin Microbial Rev, 1988; 1: 173-176. 2 Moellering RC, Jr . The Enterococci: an enigma and a continuing therapeutic challenge. Eur J Clin Microbial Infect Dis, 1990; 9: 73-74 . 3 Murray BE, Mederski-Samoraj B. Transferable betalactamase: a new mechanism for in vitro penicillin resistance in Streptococcus faecalis. J Clin Invest, 1983; 72 : 1168-1171. • Thauvin-Eliopoulos C, Rice LB, Eliopoulos GM, Moellering RC. Efficacy of oxacillin and ampicillin-sulbactam combination in experimental endocarditis caused by a beta-lactamase-hyperproducing Staphylococcus aureus. Antimicrob Agents Chemother, 1990; 34: 728-732. ' Fontana R. Leading article. Penicillin-binding proteins and the intrinsic resistance to beta-lactams in Gram-positive cocci. J Antimicrob Chemother, 1985; 16: 409-417 . 6 Fontana R, Grossato A, Rossi L, Cheng YR, Satta G . Transition from resistance to hypersusceptibility to beta-lactam antibiotics associated with a loss of a low-affinity penicillin-binding protein in a Streptococcus faecium mutant highly resistant to penicillin . Antimicrob Agents Chemother, 1985; 28 : 678-683 . 'Bertram MA, Bruckner DA, Youn LS . In vitro activity of HR 810, a new cephalosporin. Antimicrob Agents Chemother, 1984; 26, 277-279 . ' Predari SC, Ligozzi M, Fontana R. Genotypic identification of methicillin-resistant coagulase-negative Staphylococci by polymerase chain reaction. Antimicrob Agents Chemother, 1991; 35: 2568-2573. • Fontana R, Grossato A, Ligozzi M, Tonin EA. In vitro response to bactericidal activity of cell wall-active antibiotics does not support the general opinion that Enterococci are naturally tolerant to these antibiotics. Antimicrob Agents Chemother, 1990; 34: 1518-1522. 1 ° Fontana R, Canepari P, Satta G, Coyette J . Identification of the lethal target of benzylpenicillin in Streptococcus faecalis by in vivo penicillin binding studies . Nature, 1980; 287: 70-72. 11 McDougal LK, Thornsberry C . The role of beta-lactamase in staphylococcal resistance to penicillinase-resistant penicillins and cephalosporins. ] Clin Microbial, 1986; 23 : 832-839. "Lewis CM, Zeruos MJ. Clinical manifestations of enterococcal infections . Eur] Clin Microbial Infect Dis, 1990; 9: 111-117. " Schoenknecht FD, Sabath LD, Thornsberry C. Susceptibility tests: special tests. In: Manual of Clinical Microbiology, 4th ed., EH Lennette, A Balows, WJ Hausler, Jr, and HJ Shadomy (eds.) Washington, D .C ., USA, American Society for Microbiology, 1985: 1000-1008.