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In-vitro Activity of Penicillin G plus Sulbactam in Comparison with other ß-lactamase Inhibitor Combinations and Oxacillin against Staphylococci
Zbl. Bakt. 284, 297-301 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Short Communication
In-vitro Activity of Penicillin G plus Sulbactam in Comparison with other ~-lactamase Inhibitor Combinations and Oxacillin against Staphylococci * BERND JANSEW, F. PERDREAU-REMINGTON2 , and G.PULVERER Institute of Medical Microbiology, University of Cologne, Goldenfelsstrage 21, D-50935 Cologne, Germany (Head: Univ.-Prof. Dr. Dr. h. c. C. Pulverer)
Summary The in-vitro activity of penicillin G in combination with the ~-lactamase inhibitor, sulbactam, against penicillin-sensitive S. aureus (n = 10) and penicillin-resistant, methicillinsensitive S. aureus (n =69) and S. epidermidis (n =20) was tested in comparison with ampicillinlsulbactam, amoxicillinlclavulanic acid, piperacillinltazobactam and oxacillin. The combination of penicillin G plus sulbactam was found to lead to MIC values for ~-lacta mase producing staphylococci comparable to those for penicillin-sensitive staphylococci, with MIC 90 values between::; 0,03 mg/L and 0.06 mg/L. Introduction Penicillin G is still the drug of choice for Staphylococcus aureus infections due to penicillin-sensible organisms. However, penicillinase is elaborated by approximately 80% of community isolates of Staphylococcus aureus and by 70-80% of nosocomial isolates. Therefore, treatment is usually done by penicillinase-stable drugs such as dicloxacillin and flucloxacillin or by a combination of penicillins with ~-lactamase inhibitors such as ampicillinlsulbactam, amoxicillinlclavulanic acid or piperacillinltazobactam. Staphylococcus epidermidis is a leading nosocomial pathogen and its resistance towards penicillins and oxacillin is even higher than that of Staphylococcus aureus (1, 4). Several mechanisms of resistance to ~-lactam antibiotics have been de-
* Dedicated to Prof. Dr. H. Brandis on the occasion of his 80 th birthday. Present address: Univ.-Prof. Dr. Dr. B. Jansen, Head Dept. of Hygiene, Hochhaus am
1
Augustusplatz, Johannes Gutenberg-Universitat, D-55131 Mainz, Germany. 2 Present address: Prof. Dr. F. Perdreau-Remington, San Francisco General Hospital, Epi Center Epidemiology Prevention and Interventions, Dept of Medicine 5H-22, 1001 Potrero Ave., Building 100, San Francisco, CA 94110 USA.
298
B. Jansen, F. Perdreau-Remington, and G. Pulverer
scribed such as the production of a plasmid-encoded ~-lactamase and the synthesis of an additional penicillin-binding protein (PBP 2a) (6). Recently, strains have emerged with so-called borderline resistance to methicillin, probably due to a hyperproduction of ~-lactamase mimicking in vitro resistance to methicillin despite susceptibility to oxacillin and ~-lactamase inhibitors in vivo (5). Whereas methicillin-resistant staphylococci are considered to be resistant in vitro and in vivo to all ~-lactam antibiotics including carbapenems and ~-lactamase inhibitor combinations, penicillinase-producing as well as borderline-resistant strains can be treated with inhibitor-protected ~-lactams. The purpose of our study was to investigate the potential in-vitro activity of penicillin G - the most active antistaphylococcal drug - in combination with the ~-lactamase inhibitor, sulbactam, and to compare it with oxacillin and other B-lactamase inhibitor combinations such as ampicillin/sulbactam, amoxicillinlclavulanic acid and piperacillinltazobactam.
Materials and Methods Seventy-nine Staphylococcus aureus isolates were recovered from postoperative wound exudates, blood cultures, bronchoalveolar lavages, tracheal aspirates and infected prosthetic devices. They represented penicillin-sensitive (PSSA, n = 10) and penicillin-resistant but methicillin-sensitive isolates (MSSA, n =69). Staphylococcus epidermidis strains had been collected from catheter-related infections and were penicillin-resistant but methicillin-sensitive (MSSE, n = 20). Identification of Staphylococcus aureus was determined by demonstration of clumping factor and/or coagulase activity. Staphylococcus epidermidis was identified according to the Kloos/Schleifer scheme (3). Antimicrobial reference powders were kindly provided by the following manufacturers: piperacillinltazobactam (Lederle GmbH, Germany); amoxicillinlclavulanic acid, oxacillin (Smith Kline Beecham, Germany); ampicillinlsulbactam (Pfizer, Germany); penicillin G (Griinenthal, Germany). Antibiotic discs containing oxacillin (5!tg), ampicillinlsulbactam (20/10 !tg), amoxicillinl clavulanic acid (20110 !tg), piperacillinltazobactam (30110 !tg) were obtained from Oxoid, Germany. Discs with penicillin Glsulbactam (lOIS !tg) were provided by Pfizer, Germany. Staphylococcus aureus control strain ATCC 25923 was used for disc diffusion and agar dilution testing. Disc diffusion and agar dilution tests were carried out under standard conditions according to DIN standard on Mueller-Hinton agar (Oxoid). In the case of MIC testing of the inhibitor combinations, concentrations of the B-lactamase inhibitors were kept constant and were as follows: sulbactam, 10 mg/L; clavulanic acid, 2,5 mg/L and tazobactam, 4 mg/L. For the agar dilution test, a final bacterial inoculum of approximately 5 X 104 cfu/mL was plated using a multipoint inoculator. Methicillin resistance was determined with oxacillin on Mueller-Hinton agar (Difco) supplemented with 2% NaCI and incubated at 30°C for 24-48 h. Methicillin-resistant organisms were excluded from the study.
Results and Discussion Table 1 shows the MIC distribution of all antibiotics tested for the Staphylococcus aureus strains (penicillin-sensitive and penicillin-resistant ones). It is obvious that the MIC values for the combination of penicillin G and sulbactam are in the susceptible range of penicillin G alone. They are lower than those of amoxicillin/clavulanic acid and ampicillin/sulbactam and much lower than for piperacillinltazobactam and oxa-
In-vitro Activity of Penicillin G plus Sulbactam
299
Table 1. MIC values of all antibiotics tested for penicillin-sensitive (PSSA) and penicillin-resistant (MSSA) Staphylococcus aureus
Staphylococcus aureus (PSSA) n =10 MIC (mgIL)
~0.031
Penicillin G + sulbactam
10
0.06
0.125 0.25
0.5
1
2
MIC so
MIC 90
~0.031
~0.031
Ampicillin + sulbactam
3
7
0.125
0.125
Amoxicillin + clavulanic acid
8
2
0.06
0.125
1
1
Piperacillin + tazobactam
1
Oxacillin
2
5
0.25
0.5
1
9
0.5
0.5
MIC so
MIC 90
Staphylococcus aureus (MSSA) n = 69 MIC (mg/L)
~0.031
0.06
Penicillin G + sulbactam
44
25
0.125 0.25
11
36
21
Amoxicillin + clavulanic acid
12
48
9
4
13
26
12
13
30
Oxacillin
9
1
2
~
Ampicillin + sulbactam
Piperacillin + tazobactam
0.5
1
0.031
0.06
0.125
0.25
0.125
0.25
17
0.25
0.5
14
0.25
0.5
cillin. Although no breakpoints have been established for inhibitor combinations in the sensitivity testing of staphylococci * and despite the fact that testing with the agardilution method might lead to an increased susceptibility of ~-lactamase producing staphylococci against ~-lactam antibiotics, the results clearly show that combining penicillin G with sulbactam results in MICs resembling those of penicillin-sensitive strains. This holds true also for methicillin-sensitive, penicillin-resistant Staphylococcus epidermidis strains (Table 2). Table 3 shows the results of the agar disc diffusion test: The range of the inhibition zone diameters for penicillin-resistant Staphylococcus au reus and Staphylococcus epidermidis lies between 21-42 and 22-50 mm, respectively.
* In case of ~-lactamJ~-lactamase inhibitor combinations, it is recommended to use the results of oxacillin testing for interpretation.
300
B. Jansen, F. Perdreau-Remington, and G. Pulverer
Table 2. MIC values of all antibiotics tested for penicillin-resistant Staphylococcus epidermidis Staphylococcus epidermidis (MSSE) n = 20
MIC (mglL)
:=; 0.031
Penicillin G + sulbactam
14
4
Ampicillin + sulbactam
3
12
0.06
Amoxicillin + clavulanic acid
17
Piperacillin + tazobactam
2
Oxacillin
0.125 0.25
0.5
1
2
MIC so
MIC 90
2
:=;0.031
0.06
5
0.06
0.125
0.06
0.125
0.25
0.5
0.125
0.25
2 7
5
13
5
5
1
Table 3. Inhibition zone diameters of all antibiotics tested for Staphylococcus aureus and Staphylococcus epidermidis Range of inhibition zone diameters (mm)
S. aureus (PSSA) n= 10 S.aureus (MSSA) n=69 S. epidermidis (MSSE) n=20
Penicillin GI sulbactam
Ampicillin! sulbactam
Amoxicillin! Piperacillin! clavulanic acid tazobactam
Oxacillin
43-47
38-43
38-45
35-40
27-36
21-42
21-29
25-40
20-36
20-35
22-50
23-45
30-47
23-42
27-40
Breakpoints
s: =:::29mm'
(DIN)
r: <29mm
2
s:=:::16mm r: < 16mm
s = sensible; r = resistant. 1 Breakpoints for penicillin G/staphylococci. 2 No breakpoints for the testing of staphylococci established.
Concerning the breakpoints of penicillin G for staphylococci of 29 mm, some of the strains should be considered as resistant to penicillin G and sulbactam. However, also other authors found lower agar disc diffusion breakpoints for ~-lactamase inhibitor combinations, most probably due to a low inhibitor concentration in the medium (2). In conclusion, the combination of penicillin G and sulbactam leads to MIC values for ~-lactamase producing staphylococci comparable to those for penicillin-sensitive
In-vitro Activity of Penicillin G plus Sulbactam
301
staphylococci. Because of the low price and the excellent intrinsic activity of penicillin G against staphylococci, such a combination could be of great benefit in the treatment of (severe) staphylococcal infections and should be further evaluated in clinical trials. A further advantage of a treatment with penicillinlsulbactam would be the narrow spectrum of the drug combination, thus minimizing biological side-effects and problems associated with the development of resistance.
References 1. Banerjee, S. N., T. G. Emori, D. H. Culver et al.: Secular trends in nosocomial primary bloodstream infections in the United States, 1980-1989. National Nosocomial Infections Surveillance System Am. J. Med. 91 (1991) 865-895 2. Grimm, H., M. Springsklee, A. F. Schmalreck, A. Bauernfeind, J. Focht, W. Opferkuch, and H. Werner: Bewertungsgrenzen fur die Empfindlichkeitsprufung bakterieller Erreger gegen MezlocillinlSulbactam, PiperacillinlSulbactam, CefotaximlSulbactam und CefoperazonlSulbactam im Agardiffusionstest nach DIN 58940. Zeitschrift fUr antimikrobielle antineoplastische Chemotherapie 11 (1993) 39-49 3. Kloos, W. E. and K. H. Schleifer: Simplified scheme for routine identification of human Staphylococcus species. Journal of Clinical Microbiology 1 (1975) 82-88 4. Martin, M. A., M. A. Pfaller, and R. P. Wenzel: Coagulase-negative staphylococcal bacteremia. Mortality and hospital stay. Annals ofInternal Medicine 110 (1989) 9-16 5. McDougal, L. K. and C. Thornsberry: The role of ~-lactamase in staphylococcal resistance to penicillinase-resistant penicillins and cephalosporines. Journal of Clinical Microbiology 23 (1986) 832-839 6. Sabath, L. D.: Mechanisms of resistance to beta-lactam antibiotics in strains of Staphylococcus aureus. Annals of Internal Medicine 97 (1982) 339-344
Univ.-Prof. Dr. Dr. B.Jansen, Head Dept. of Hygiene, Hochhaus am Augustusplatz, Johannes-Gutenberg-Universitiit, D-55131 Mainz, Germany
Short Communication
In-vitro Activity of Penicillin G plus Sulbactam in Comparison with other ~-lactamase Inhibitor Combinations and Oxacillin against Staphylococci * BERND JANSEW, F. PERDREAU-REMINGTON2 , and G.PULVERER Institute of Medical Microbiology, University of Cologne, Goldenfelsstrage 21, D-50935 Cologne, Germany (Head: Univ.-Prof. Dr. Dr. h. c. C. Pulverer)
Summary The in-vitro activity of penicillin G in combination with the ~-lactamase inhibitor, sulbactam, against penicillin-sensitive S. aureus (n = 10) and penicillin-resistant, methicillinsensitive S. aureus (n =69) and S. epidermidis (n =20) was tested in comparison with ampicillinlsulbactam, amoxicillinlclavulanic acid, piperacillinltazobactam and oxacillin. The combination of penicillin G plus sulbactam was found to lead to MIC values for ~-lacta mase producing staphylococci comparable to those for penicillin-sensitive staphylococci, with MIC 90 values between::; 0,03 mg/L and 0.06 mg/L. Introduction Penicillin G is still the drug of choice for Staphylococcus aureus infections due to penicillin-sensible organisms. However, penicillinase is elaborated by approximately 80% of community isolates of Staphylococcus aureus and by 70-80% of nosocomial isolates. Therefore, treatment is usually done by penicillinase-stable drugs such as dicloxacillin and flucloxacillin or by a combination of penicillins with ~-lactamase inhibitors such as ampicillinlsulbactam, amoxicillinlclavulanic acid or piperacillinltazobactam. Staphylococcus epidermidis is a leading nosocomial pathogen and its resistance towards penicillins and oxacillin is even higher than that of Staphylococcus aureus (1, 4). Several mechanisms of resistance to ~-lactam antibiotics have been de-
* Dedicated to Prof. Dr. H. Brandis on the occasion of his 80 th birthday. Present address: Univ.-Prof. Dr. Dr. B. Jansen, Head Dept. of Hygiene, Hochhaus am
1
Augustusplatz, Johannes Gutenberg-Universitat, D-55131 Mainz, Germany. 2 Present address: Prof. Dr. F. Perdreau-Remington, San Francisco General Hospital, Epi Center Epidemiology Prevention and Interventions, Dept of Medicine 5H-22, 1001 Potrero Ave., Building 100, San Francisco, CA 94110 USA.
298
B. Jansen, F. Perdreau-Remington, and G. Pulverer
scribed such as the production of a plasmid-encoded ~-lactamase and the synthesis of an additional penicillin-binding protein (PBP 2a) (6). Recently, strains have emerged with so-called borderline resistance to methicillin, probably due to a hyperproduction of ~-lactamase mimicking in vitro resistance to methicillin despite susceptibility to oxacillin and ~-lactamase inhibitors in vivo (5). Whereas methicillin-resistant staphylococci are considered to be resistant in vitro and in vivo to all ~-lactam antibiotics including carbapenems and ~-lactamase inhibitor combinations, penicillinase-producing as well as borderline-resistant strains can be treated with inhibitor-protected ~-lactams. The purpose of our study was to investigate the potential in-vitro activity of penicillin G - the most active antistaphylococcal drug - in combination with the ~-lactamase inhibitor, sulbactam, and to compare it with oxacillin and other B-lactamase inhibitor combinations such as ampicillin/sulbactam, amoxicillinlclavulanic acid and piperacillinltazobactam.
Materials and Methods Seventy-nine Staphylococcus aureus isolates were recovered from postoperative wound exudates, blood cultures, bronchoalveolar lavages, tracheal aspirates and infected prosthetic devices. They represented penicillin-sensitive (PSSA, n = 10) and penicillin-resistant but methicillin-sensitive isolates (MSSA, n =69). Staphylococcus epidermidis strains had been collected from catheter-related infections and were penicillin-resistant but methicillin-sensitive (MSSE, n = 20). Identification of Staphylococcus aureus was determined by demonstration of clumping factor and/or coagulase activity. Staphylococcus epidermidis was identified according to the Kloos/Schleifer scheme (3). Antimicrobial reference powders were kindly provided by the following manufacturers: piperacillinltazobactam (Lederle GmbH, Germany); amoxicillinlclavulanic acid, oxacillin (Smith Kline Beecham, Germany); ampicillinlsulbactam (Pfizer, Germany); penicillin G (Griinenthal, Germany). Antibiotic discs containing oxacillin (5!tg), ampicillinlsulbactam (20/10 !tg), amoxicillinl clavulanic acid (20110 !tg), piperacillinltazobactam (30110 !tg) were obtained from Oxoid, Germany. Discs with penicillin Glsulbactam (lOIS !tg) were provided by Pfizer, Germany. Staphylococcus aureus control strain ATCC 25923 was used for disc diffusion and agar dilution testing. Disc diffusion and agar dilution tests were carried out under standard conditions according to DIN standard on Mueller-Hinton agar (Oxoid). In the case of MIC testing of the inhibitor combinations, concentrations of the B-lactamase inhibitors were kept constant and were as follows: sulbactam, 10 mg/L; clavulanic acid, 2,5 mg/L and tazobactam, 4 mg/L. For the agar dilution test, a final bacterial inoculum of approximately 5 X 104 cfu/mL was plated using a multipoint inoculator. Methicillin resistance was determined with oxacillin on Mueller-Hinton agar (Difco) supplemented with 2% NaCI and incubated at 30°C for 24-48 h. Methicillin-resistant organisms were excluded from the study.
Results and Discussion Table 1 shows the MIC distribution of all antibiotics tested for the Staphylococcus aureus strains (penicillin-sensitive and penicillin-resistant ones). It is obvious that the MIC values for the combination of penicillin G and sulbactam are in the susceptible range of penicillin G alone. They are lower than those of amoxicillin/clavulanic acid and ampicillin/sulbactam and much lower than for piperacillinltazobactam and oxa-
In-vitro Activity of Penicillin G plus Sulbactam
299
Table 1. MIC values of all antibiotics tested for penicillin-sensitive (PSSA) and penicillin-resistant (MSSA) Staphylococcus aureus
Staphylococcus aureus (PSSA) n =10 MIC (mgIL)
~0.031
Penicillin G + sulbactam
10
0.06
0.125 0.25
0.5
1
2
MIC so
MIC 90
~0.031
~0.031
Ampicillin + sulbactam
3
7
0.125
0.125
Amoxicillin + clavulanic acid
8
2
0.06
0.125
1
1
Piperacillin + tazobactam
1
Oxacillin
2
5
0.25
0.5
1
9
0.5
0.5
MIC so
MIC 90
Staphylococcus aureus (MSSA) n = 69 MIC (mg/L)
~0.031
0.06
Penicillin G + sulbactam
44
25
0.125 0.25
11
36
21
Amoxicillin + clavulanic acid
12
48
9
4
13
26
12
13
30
Oxacillin
9
1
2
~
Ampicillin + sulbactam
Piperacillin + tazobactam
0.5
1
0.031
0.06
0.125
0.25
0.125
0.25
17
0.25
0.5
14
0.25
0.5
cillin. Although no breakpoints have been established for inhibitor combinations in the sensitivity testing of staphylococci * and despite the fact that testing with the agardilution method might lead to an increased susceptibility of ~-lactamase producing staphylococci against ~-lactam antibiotics, the results clearly show that combining penicillin G with sulbactam results in MICs resembling those of penicillin-sensitive strains. This holds true also for methicillin-sensitive, penicillin-resistant Staphylococcus epidermidis strains (Table 2). Table 3 shows the results of the agar disc diffusion test: The range of the inhibition zone diameters for penicillin-resistant Staphylococcus au reus and Staphylococcus epidermidis lies between 21-42 and 22-50 mm, respectively.
* In case of ~-lactamJ~-lactamase inhibitor combinations, it is recommended to use the results of oxacillin testing for interpretation.
300
B. Jansen, F. Perdreau-Remington, and G. Pulverer
Table 2. MIC values of all antibiotics tested for penicillin-resistant Staphylococcus epidermidis Staphylococcus epidermidis (MSSE) n = 20
MIC (mglL)
:=; 0.031
Penicillin G + sulbactam
14
4
Ampicillin + sulbactam
3
12
0.06
Amoxicillin + clavulanic acid
17
Piperacillin + tazobactam
2
Oxacillin
0.125 0.25
0.5
1
2
MIC so
MIC 90
2
:=;0.031
0.06
5
0.06
0.125
0.06
0.125
0.25
0.5
0.125
0.25
2 7
5
13
5
5
1
Table 3. Inhibition zone diameters of all antibiotics tested for Staphylococcus aureus and Staphylococcus epidermidis Range of inhibition zone diameters (mm)
S. aureus (PSSA) n= 10 S.aureus (MSSA) n=69 S. epidermidis (MSSE) n=20
Penicillin GI sulbactam
Ampicillin! sulbactam
Amoxicillin! Piperacillin! clavulanic acid tazobactam
Oxacillin
43-47
38-43
38-45
35-40
27-36
21-42
21-29
25-40
20-36
20-35
22-50
23-45
30-47
23-42
27-40
Breakpoints
s: =:::29mm'
(DIN)
r: <29mm
2
s:=:::16mm r: < 16mm
s = sensible; r = resistant. 1 Breakpoints for penicillin G/staphylococci. 2 No breakpoints for the testing of staphylococci established.
Concerning the breakpoints of penicillin G for staphylococci of 29 mm, some of the strains should be considered as resistant to penicillin G and sulbactam. However, also other authors found lower agar disc diffusion breakpoints for ~-lactamase inhibitor combinations, most probably due to a low inhibitor concentration in the medium (2). In conclusion, the combination of penicillin G and sulbactam leads to MIC values for ~-lactamase producing staphylococci comparable to those for penicillin-sensitive
In-vitro Activity of Penicillin G plus Sulbactam
301
staphylococci. Because of the low price and the excellent intrinsic activity of penicillin G against staphylococci, such a combination could be of great benefit in the treatment of (severe) staphylococcal infections and should be further evaluated in clinical trials. A further advantage of a treatment with penicillinlsulbactam would be the narrow spectrum of the drug combination, thus minimizing biological side-effects and problems associated with the development of resistance.
References 1. Banerjee, S. N., T. G. Emori, D. H. Culver et al.: Secular trends in nosocomial primary bloodstream infections in the United States, 1980-1989. National Nosocomial Infections Surveillance System Am. J. Med. 91 (1991) 865-895 2. Grimm, H., M. Springsklee, A. F. Schmalreck, A. Bauernfeind, J. Focht, W. Opferkuch, and H. Werner: Bewertungsgrenzen fur die Empfindlichkeitsprufung bakterieller Erreger gegen MezlocillinlSulbactam, PiperacillinlSulbactam, CefotaximlSulbactam und CefoperazonlSulbactam im Agardiffusionstest nach DIN 58940. Zeitschrift fUr antimikrobielle antineoplastische Chemotherapie 11 (1993) 39-49 3. Kloos, W. E. and K. H. Schleifer: Simplified scheme for routine identification of human Staphylococcus species. Journal of Clinical Microbiology 1 (1975) 82-88 4. Martin, M. A., M. A. Pfaller, and R. P. Wenzel: Coagulase-negative staphylococcal bacteremia. Mortality and hospital stay. Annals ofInternal Medicine 110 (1989) 9-16 5. McDougal, L. K. and C. Thornsberry: The role of ~-lactamase in staphylococcal resistance to penicillinase-resistant penicillins and cephalosporines. Journal of Clinical Microbiology 23 (1986) 832-839 6. Sabath, L. D.: Mechanisms of resistance to beta-lactam antibiotics in strains of Staphylococcus aureus. Annals of Internal Medicine 97 (1982) 339-344
Univ.-Prof. Dr. Dr. B.Jansen, Head Dept. of Hygiene, Hochhaus am Augustusplatz, Johannes-Gutenberg-Universitiit, D-55131 Mainz, Germany