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tazobactam
Pathology (1996), 28, pp. 167-172
AN EVALUATION OF THE IN VITRO ACTIVITY OF PIPERACILLIN/TAZOBACTAM D. DALEY*, L. MULGRAVE'~, R. MUNRO*, S. NEVILLE*, H. SMITH~ AND
W. DIMECH$
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*Department of Microbiology and Infectious Diseases, South Western Area Pathology Service, Liverpool Hospital, Liverpool, NSW; ?Department of Microbiology; WA Centre for Pathology and Medical Research, Nedlands, WA; and ~:Department of Microbiology, Royal Melbourne Hospital, Melbourne, Vic
Summary
Key words: Piperaciltin, tazobactam, staphylococci.
Tazobactam is a new, irreversible inhibitor of bacterial betalactamases of staphylococci, piasmid-mediated beta-lactamases of the TEM and SHV types found in Escherichia coil and Klebsielta species and beta-lactamases of anerobes such as Bacteroides species. Its combination with piperacillin, a broad spectrum ureido-penicillin, would be expected to improve the activity of piperacillin against staphylococci, TEM and SHV betalactamase producing Gram negative bacteria and anerobes. Minimal inhibitory concentrations (MIC) of piperacillin/ tazobactam were determined for 1952 individual patient isolates of Gram positive and negative bacteria causing significant infections and compared with MIC values for cefotaxime, ceftazidime, ciprofloxacin, imipenem, ticarcillin/clavulanic acid. MlCs were determined by agar dilution (NCCLS 1990 and 1992). Piperacillin/tazobactam had excellent activity against methicillin susceptible staphylococci, Streptococcus pneumoniae, Haemophilus influenza& enterococci and organisms of the Bacteroides fragilis group. It was also active against the majority of Enterobacteriaceae and Pseudomonas aeruginosa isolates tested. It was not active against extended spectrum beta-lactamase (ESBL) producing Klebsiella species and some high level TEM and SHV beta-lactamase producing E. coil and Klebsiella species. Activity against Gram negative organisms capable of producing chromosomally mediated beta-lactamases was good, since in most organisms tested, the enzymes were not induced in sufficient quantities to cause antibiotic resistance. However some Enterobacter species were derepressed hyperproducing mutants; these isolates showed resistance to piperacillin/tazobactam since tazobactam does not inhibit these Class I beta tactamases. Activity was superior to ticarcillin/ clavulanic acid for Gram negative rods. lmipenem was the most active agent against ESBL producing Klebsiella species. Piperacillin/tazobactam has a suitable spectrum of activity in vitro to suggest its use in monotherapy of mixed anerobic infections, mixed respiratory infections such as aspiration pneumonia and, in combination with an aminoglycoside, it would provide Gram positive as well as Gram negative cover of febrile episodes in immunosuppressed patients.
Accepted 12 December 1995
INTRODUCTION
Beta-lactamase production is one of the most important mechanisms of antibiotic resistance in bacteria. The large number of different beta-lactamases produced by Gram positive and negative bacteria are either plasmid- or transposon-mediated, and thus capable of dissemination to other bacteria, or determined by chromosomal DNA. Among Gram negative bacteria and staphylococci, plasmid-mediated beta-lactamases are widespread and of many different types. In Gram negative bacteria the common ones are the TEM-1, TEM-2 and SHV-1 enzymes which hydrolyse ampicillin, first generation cephalosporins and other penicillins. TEM-1 and related enzymes are now widespread in enterobacteria and are present in the majority of Escherichia coti. 1-3 Their wide distribution and broad spectrum of activity have reduced the clinical usefulness of ampicillin and first generation cephalosporins and monobactams. Organisms with higher levels of enzyme production are also resistant to extended spectrum penicillins such as ticarcillin and piperacillin. Recently point mutations in TEM-1, TEM2 and SHV-1 enzymes have resulted in more extended spectrum beta-lactamases which hydrolyse third and fourth generation cephalosporins. First described in Ktebsiella species in Germany,4 these enzymes are now found worldwide especially in E. coli and KlebsielIa species. 3 In addition, hyper-production of TEM-1 and SHV-1 enzymes has resulted in increased resistance to combinations of betaIactam antibiotics with beta-lactam inhibitors) '6 The most important chromosomalty-determined betalactamases are the class 1 enzymes which occur in enterobacteria and Pseudomonas aeruginosa; they hydrolyse a wide range of cephalosporins and broad spectrum penicillins. Most Gram negative bacteria possess the chromosomal information to produce beta-lactamases but production is in insufficient quantities to cause antibiotic resistance. In Enterobacter, Citrobacter, Serratia, Proteus
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168
Pathology (1996), 28, May
DALEYetaL
vulgaris, Morganella, Providencia species, and Pseudomonas aeruginosa the enzymes are inducible but these organisms remain susceptible to many new broad spectrum penicillins and cephalosporins since the antibiotics are poor inducers. However in all these species there are mutant subpopulations which constitutively produce class 1 enzymes at high levels] Therefore the use of beta-lactam antibiotics should be avoided in infections caused by organisms producing inducible beta-lactamases since selection of resistant mutants is possible with resultant treatment failure. ~ Piperacillin is a broad spectrum ureido-penicillin which is inactivated by staphylococcal beta-lactamases, by some of the TEM and SHV enzymes of Gram negative bacteria and, although inactivated by class 1 enzymes, retains in vittv activity against inducible enzyme producing organisms because in itself it is a poor inducer of beta-lactamases. However organisms constitutively producing class 1 enzymes are not susceptible to piperacillin. In addition, piperacillin is inactivated by" beta-lactamases of Bacteroides species. Tazobactam is a penicillanic acid sulphone which can inhibit beta-lactamases of staphylococci, some of the common plasmid-mediated TEM and SHV beta-lactamases of E. coli, H. influenzae and KIebsiella species and betalactamases of anerobes such as Bacteroides species. 922 It does not inhibit class 1 chromosomally-determined betalactamases found in Enterobacter, Citrobacter, Serratia, Proteus vulgaris, Pseudomonas, and similar species. The combination of tazobactam with piperaciltin would be expected to restore the activity of piperacillin against those organisms producing beta-lactamases inhibited by tazobactam. The present study was undertaken to investigate the efficacy of piperacillin/tazobactam in vitro against 1,952 isolates causing significant infections in Australian hospitals and to compare its activity with ceftazidime, cefotaxime, imipenem, ciprofloxacin and ticarcillin/clavulanic acid.
MATERIALS AND METHODS Organism~ A total of 1,952 clinical isolates were collected and tested in 3 Australian states. The strains represent a broad cross section of mostly hospital acquired isolates that were found to be causing significant sepsis. From New South Wales, the South Western Area Pathology Service tested 530 isolates from one tertiary referral hospital and 5 smaller hospitals with a total of 1,500 beds. The Microbiology Department of the Royal Melboume Hospital, Victoria and the 660 bed Sir Charles Gairdner Hospital, Perth, Western Australia, both ternary teaching hospitals, tested 459 and 940 strains respectively. The latter collection included 33 extended-spectrum beta-lactamase producing strains collected since they first appeared in 1989 from hospitals in all Australian mainland states. All were unique patient isolates from clinically significant infections. In particular, isolates of coagulase negative staphylococci and enterococci were only included if from normally sterile sites or documented intravascular infections, Similarly, isolates of S. pneumoniae and H. influenzae were mostly from sterile sites. Respiratory isolates were only included from definite cases of pneumonia. Identification of organisms was as follows: Gram negative rods were identified using either Vitek AlVlS or the ATB32E and ATB32GN systems (Bit Merieux SA, 69280 Marcy l'Etoile, France). Enterococci were identified using conventional biochemical media according to Facklam's criteria ~3 and S. pneumoniae on the basis of optochin susceptibility. H. influenzae was identified on the basis of X and V requirements.
TABLE 1 Organisms included in the study Organism
Escherichia coli Klebsiella spp. total ESBL 1 Klebsiella spp. Enterobacter spp. total Cittvbacter/Serratia spp. total Morganella/Pmteus/Providencia spp. total Proteus mirabilis Pseudomonas aeruginosa Acinetobacter calcoaceticus Staphylococcus aureus Coagulase negative Staphylococcus spp. Streptococcus pneumoniae Enterococcus spp. total Haemophilus influenzae Bacteroides fragitis group
Total
Number tested 528 224 44 142 84 45 104 88 40 433 28 45 109 59 23 1952
Staphylococci were identified with tube and slide tests for coagulase, DNAse and phosphatase production, anerobic fermentation of mannitol and hydrolysis with Tween 80. Anerobes were identified using the ATB32A system (Bit Merieux SA, 69280 Marcy l'Etoile, France).
Antibiotic susceptibility testing Minimal inhibitory concentrations (MIC) for all organisms except H. influenzae were determined using the agar dilution methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS)J 4'15 The only variation to that method was the incorporation of p-(4-nitrophenyt)-glycerol 43 mg/L (PNPG) into all plates except those involving specific organisms such as S. pneumoniae and anerobes. PNPG prevents swarming of Proteus species and has been previously shown not to influence MIC results. 16 Antibiotics tested were obtained from the manufacturer as pure substances of known potency. All antibiotic-containing agar plates were used within 72 hrs of preparation, with the exception of imipenem which was used on the day of preparation. Doubling dilutions were prepared for the following antibiotics: piperacillin 0.004-256 mg/L with a fixed concentration of tazobactam of 4 mg/L in each plate; cefotaxime 0.06-128 mg/L; imipenem 0.06-64 mg/L; ceftazidime 0.06-64 mg/L; ticarcillin 0.06-256 mg/L with a fixed concentration of clavulanic acid of 2 mg/L in each plate and ciprofloxacin 0.06-8 mg/L. MICs for H. influenzae isolates were determined using the E test (AB Biodisk, Pyramidvagen 7, 5-17136 Solina, Sweden)) 7
tntra-laboratory reproducibility Each laboratory tested control organisms as stipulated by the NCCLS method with each antibiotic tested. In addition every 25th organism tested at each centre was re-tested at the other centres. Organisms exchanged for re-testing were designed to occur randomly, but also to represent each group of test organisms adequately. The MIC to inhibit 50% (MIC50) and 90% (MIC90) of isolates was calculated as well as the % resistance at breakpoint concentrations recommended by the NCCLS.
RESULTS All control organisms tested gave satisfactory results according to NCCLS guidelines. A total of 28 organisms were exchanged between testing laboratories. All MIC results were within one doubling dilution of the MIC determined by the referring laboratory. These results indicated adequate intra-laboratory reproducibility and allowed for pooling of results. Table 1 summarizes the identification and numbers of organisms tested. Table 2 documents MIC50 and MIC90 determinations. Table 3 summarizes the per cent resistance at breakpoints recommended by the
AN EVALUATIONOF THE IN VITROACTIVITYOF PIPERACILLIN/TAZOBACTAM TABLE2 Minimal inhibitory concentrations (MIC) for 1,952 clinically significant isolates Organism number)
Range
MIC50
MIC90
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E. coli (528) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Ktebsiella spp. (180) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Klebsiella spp. (ESBL 44) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Enterobacter spp. (142) piperacillin/tazobactam ticarcillirdclavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Citrobacter/Serratia spp, (84) piperacitlin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin
0.25 - 128.0 0.25 ->256.0 0.06 2.0 0.03 - 16.0 0.03 8.0 0.0080.5
1.0 2.0 0.06 0.06 0.06 0.015
0.25 ->256.0 0.5 -->256.0 0.06 2.0 0.03 8,0 0.03 - 16.0 0.0081.0
1.0 1.0 0.06 0.06 0.06 0.03
1.0 ->256.0 8.0 ->256.0 0.1251.0 4.0 - >64.0 0.03 - 16.0 0.0168.0
8.0 32.0 0.125 4.0 4.0 t.0
64.0 128.0 0.25 64.0 16.0 2.0
0.004- 128.0 0.25 ->256.0 0.1254.0 0.03 - >64.0 0.03 ->128.0 0.0082.0
2.0 4.0 0.125 0.25 0.125 0.03
16.0 128.0 1.0 32.0 32.0 0.O6
0.25 - 256.0 0.5 ->256.0 0.06 8.0 0.03 - >64.0 0.03 ->128.0 0.0081.0
2.0 4.0 0.25 0.25 0.125 0.06
8.0 64.0 2.0 1.0 8.0 0.12
2.0 8.0 2.0 16.0 0.5 0.5
2.0 32.0 0.25 0.25 0.25 0.03 4.0 4.0 0.25 0.25 0.125 0.06
169
TABLE 2 Continued Organism (number) Coagulase negative Staphylococcal (28) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin
Range
MIC50
0.125- 256.0 0.125->256.0 0 . 0 6 - 64.0 4.0 - >64.0 0.25 ->128.0 0.1250.5
0.5 1.0 0.06 8.0 2,0 0,25
0.004- 0.25 0.06 4.0 0,06 0.06 2.0 0.06 0.25 2.0
0.001 0.25 -<0,06 0,12: -<0.06 1.0
MIC90
16.0 64.0 8.0 >64.0 >128.0 0.25
Streptococcus pneumoniae (45) piperacillin/tazobactam ticarciUin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Enterococci (109) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Haemophilus influenzae (59) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Bacteroidesfragilis gp (23) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin
0.06 ->256.0 1.0 ->256.0 0 . 0 6 - 64.0 0.06 - >64.0 0.03 ->128.0 0.125- >8.0
2.0 32,0 1.0 >64.0 64.0 1.0
<0.016-0.25 0.0471.5 0.064-- >32.0 0.0471.5 0.003 -0.047 0.003- 0.19
<0,01~ 0.12: 0.75
0.004->256.0 0.06 - 256.0 0.06 - 16.0 16.0 - >64.0 32.0 ->128.0 1.0 - > 8 . 0
0.5 4,0 0.25 64.0 64.0 4.0
0,12:
0.0E 0.01:
0.015 0.5 -<0.06 0.25 -<0.06 2.0 4.0 64.0 2.0 >64.0 >128.0 2.0 0.094 0.5 >32.0 0.25 0.032 0.016 4.0 32.0 2.0 >64.0 >128.0 >8.0
Morganella/Proteus/ Providencia spp, (45) piperacillirdtazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Proteus mirabilis (104) piperacillin/tazobactam ticarciUin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Pseudomonas aeruginosa (88) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Acinetobacter calcoaceticus (40) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Staphylococcus aureus (433) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin
0.1250.250.06 0.060.03 0.008-
4.0 32.0 2.0 32.0 8.0 8.0
0,25 1.0 1.0
0.1250.25 0.1250.03 0.030.008-
2.0 16.0 4.0 0.25 0.25 0.5
0.25 0.5 2.0 0.06 0.06 0.03
0.125
0.06 0.015
0.5 1.0 4.0 0.06 0.06 0.06
0.5 ->256.0 2.0 ->256.0 0.5 - 32.0 0.5 - 16.0 2.0 ->128,0 0 . 0 6 - >8.0
4.0 16.0 2.0 2.0 32.0 0.125
32.0 64.0 16.0 8.0 128.0 1.0
0.25 - 64.0 0.5 - 128.0 0.1251.0 0.03 - 16.0 0.125- 64.0 0.0158,0
16.0 32.0 0.25 4.0 32.0 0.5
32.0 64.0 0.5 16.0 64.0 2.0
0.125-256.0 0.25 ->256.0 0 . 0 6 - 64.0 0.5 - >64,0 0.125->128.0 0.125- >8.0
1.0 1.0 0.125 8.0 2.0 0.5
4.0 32.0 1.0 32.0 16.0 1.0
NCCLS. Breakpoints are not provided by the NCCLS for S.
pneumoniae and enterococci for any of the antibiotics tested, for ticarcillin/clavulanic acid against any Gram positive organisms and for piperacillin/tazobactam and ticarcillin/clavulanic acid against H. influenzae. Activity against Gram negative organisms A g a i n s t E. coli and Klebsiella s p e c i e s , the t w o m o s t c o m m o n G r a m n e g a t i v e o r g a n i s m g r o u p s , all antibiotics t e s t e d h a d g o o d activity w i t h l o w levels o f r e s i s t a n c e e x c e p t a m o n g E S B L p r o d u c i n g KlebsielIa s p e c i e s . T h e s e o r g a n i s m s s h o w e d characteristically h i g h levels o f resistance to b e t a - l a c t a m s w i t h the e x c e p t i o n o f i m i p e n e m . N e i t h e r t a z o b a c t a m n o r clavulanic acid i n h i b i t e d t h e s e E S B L s and the o r g a n i s m s w e r e r e s i s t a n t to b o t h p i p e r a c i l l i n / t a z o b a c t a m and ticarcillin/clavulanic acid. N i n e t e e n p e r c e n t o f o t h e r w i s e s u s c e p t i b l e E. coli and 7 % o f n o n - E S B L Klebsiella s p e c i e s w e r e r e s i s t a n t to ticarcillin/ clavulanic acid b u t o n l y 4 % o f Klebsiella s p e c i e s and 1% o f E. coIi w e r e also r e s i s t a n t to p i p e r a c i l l i n / t a z o b a c t a m . T h e s e o r g a n i s m s s h o w e d o n l y l o w levels o f r e s i s t a n c e to o t h e r c e p h a l o s p o r i n s tested, T h e antibiotic s u s c e p t i b i l i t y p a t t e r n s o f t h e s e E. coli and Klebsiella s p e c i e s are c h a r a c t e r i s t i c o f o r g a n i s m s w i t h h i g h levels o f p r o d u c t i o n o f T E M o r S H V b e t a - l a c t a m a s e s 18 and e x h i b i t i n g r e s i s t a n c e to ampicillin, a m o x y c i l l i n / c l a v u l a n i c acid, t i c a r c i l l i n a n d p i p e r a c i l l i n (results not s h o w n ) . T h e d i f f e r e n c e in the f r e q u e n c y o f
170
DALEYet al.
Pathology (1996), 28, May
TABLE3 Per cent resistance at NCCLS breakpoints Organism
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(Number) E. coli (528) Klebsiella spp. (180) KtebsielIa spp. (ESBL44) Enterobacter spp. (142) Citrobacter/Serratia spp. (84) Morganetla/Providencia/ Proteus spp. (45) P. mirabilis (104) Ps. aeruginosa (88) Acinetobacter calcoaceticus (40) S. aureus (433) Coagulase neg. Staphylococcal(28) Haemophilus influenzae (59) Bacteroidesfragilis gp (23) Breakpoints mg/L) Staphylococci Gram neg. rods Pseudomonas Anerobes
Antibiotic Ceftax
Ceftaz
Imipen
Pip/Taz
Tic/Clay
Cipro
0% 2% 16% 18% 7%
1% 0% 27% 19% 10%
0% 0% 0% 0% 1%
1% 4% 13% 11% 4%
19% 7% 70% 28% 17%
0% 0% 11% 1% 0%
0% 0% 71% 80% 11% 25% 0% 74%
11% 0% 6% 12% 18% 37% 0%
0% 0% 29% 0% 8% 14% 19% 4%
0% 0% 2% 50% 9% 12%
2% 0% 7% 60%
4%
4%
11% 0% 10% 20% 4% 0% 0%
8 8 8
8 8 8
32
8
16 64 ~32/4
16 64 128
1 1 1
Ceftax=Cefotaxime Ceftaz=Ceftazidime Imipen=Irnipenem Pip/Taz=Piperacillin/Tazobactam Tic/Clav=Ticarcillin/Clavulanicacid Cipro=Ciprofloxacin resistance between the two beta-lactam/beta-lactamase inhibitors probably reflects differing amounts of betalactamase produced and the different concentrations of inhibitor in the plates (2 mg/L of clavulanic acid versus 4 mg/L oftazobactam). The divergence in MIC50 and MIC90 among E. coli for ticarcillin/clavulanic acid (MIC50 2 rag/L, MIC90 32 rag/L) suggests a subpopulation of high level enzyme producers. Isolates of Pseudomonas aeruginosa were among the most antibiotic resistant. Resistance to imipenem, cefotaxime, ticarcillin/clavulanic acid and piperacillin/tazobactam may be due to production of the class l a m p C beta-lactamase which is not inhibited by clavulanic acid or tazobactam along with a porin mutation specific for imipenem. All Proteus mirabilis strains examined were uniformly susceptible to all six agents tested. Some strains of Enterobacter, Citrobacter and Serratia species harboured derepressed chromosomal beta-lactamases, rendering them resistant to all the beta-lactams tested including both inhibitor combinations. Pooled results showed the MICs of piperacillin/tazobactam for these isolates to be consistently lower than that for ticarcillin/clavulanic acid but only by a single doubling dilution. When the results were analysed by Australian state, two of three states recorded a four-fold difference favouring piperacillin/tazobactam. The difference in susceptibility between piperacillin/tazobactam and ticarcillin/clavulanic acid probably results from the different amounts of each inhibitor used in testing. Alternatively clavulanic acid (but not tazobactam), which is a strong inducer of class I enzymes, 19 may have facilitated destruction of ticarcillin by in vitro enzyme induction. Activity against staphylococci Data presented in the tables include both MRSA and MSSA, as the data were not analysed separately.
Piperacillin/tazobactam had very good activity against methicillin sensitive S. aureus isolates. Low levels of resistance were also seen for other antibiotics tested against these strains. All resistance to beta-lactam antibiotics seen in staphylococci occurred in methicillin resistant isolates. It is generally considered that in vitro susceptibility results for beta-lactam antibiotics are inaccurate for MRSA isolates and should be regarded as resistant to all beta-lactam antibiotics. Ciprofloxacin resistance was common among MRSA isolates. Susceptibility of coagulase negative staphylococci to beta-lactams including piperacillin/ tazobactam was generally the same as susceptibility to methicillin. Activity against S. pneumoniae and enterococci
Very low MICs were seen in all antibiotics tested against these organisms. There were no penicillin-resistant isolates of S. pneumoniae in the collections tested. A small number of enterococcal isolates were E. faecium - these were resistant to all beta-lactams tested including piperacillin/tazobactam. Broad-spectrum beta-lactam resistance in these organisms is due to mutation of penicillin-binding proteins. 2° Activity against H. influenzae
With the exception of imipenem (19% resistant), all strains tested were found to be susceptible to the remaining antibiotics tested. Activity against Bacteroides fragilis group High MICs were seen for the third generation cephalosporins tested and to ciprofloxacin. Piperacillin/tazobactam had good activity against these organisms. NCCLS breakpoints for anerobes were not available for ceftazidime and ciprofloxacin.
AN EVALUATIONOF THE IN VITRO ACT[VITY OF PIPERACILLIN/TAZOBACTAM
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DISCUSSION Antibiotic susceptibility patterns will vary in different hospitals reflecting the distribution of organisms causing infections and their resistance mechanisms. Piperacillin/ tazobactam has the potential to provide broad-spectrum activity against staphylococci, streptococci, aerobic Gram negative rods and anerobes which are resistant to piperacillin by virtue of beta-lactamase production if the enzyme can be inhibited by tazobactam, In this study piperacillin/tazobactam and imipenem were the most active antibiotics tested. While imipenem retained activity aginst ESBL producing Klebsiella species, lower levels of resistance were seen to piperacillin/tazobactam than to imipenem among Ps. aeruginosa. ESBL producing Klebsiella pneumoniae and strains of Enterobacteriaceae that hyperproduce TEM and SHV beta-lactamases are an increasing problem in Australian hospitals. ~1Although ESBL enzymes are known to be susceptible to inhibition by clavulanic acid and tazobactam, the production of these enzymes in addition to the SHV-1 and TEM-1 enzymes is tantamount to hyperproduction. HyperTEM-1 production in E. coli is a mechanism proven to overwhelm clavulanic acid at clinically achievable levels.22 This mechanism is the probable explanation for the poor activity of inhibitor combinations toward ESBL strains shown by the pooled results (MIC90 range 64-128 mg/L). A second possible explanation is the occurrence of mutants with reduced affinity of their betalactamases to the inhibitors. However such variants are rare at the present time, although this situation may change in the future with greater clinical use of inhibitor combinations.23 Because of its broad antibacterial effects, piperacillin/ tazobactam has potential as monotherapy in mixed infections and also as part of empirical therapy in immunosuppressed patients where activity against staphylococci is as necessary as Gram negative cover.24 Unless methicillin resistance is common in staphylococcal isolates piperacillin/ t~obactam can provide both Gram positive and Gram negative cover in conjunction with an aminoglycoside in these circumstances. Piperacillin/tazobactam activity against S. pneumoniae and H. influenzae also make it attractive as monotherapy for mixed respiratory infections such as aspiration pneumonia. Other mixed anerobic infections in the gastrointestinal and genitourinary systems are similar targets for piperacillin/tazobactam monotherapy. Reports of clinical trials with piperacillin/tazobactam indicate its usefulness in intra-abdominal infections, 25lower respiratory tract infection, 26 and febrile neutropenic patientsY The safety and efficacy profile of piperacillin/ tazobactam in over 1,200 patients was excellent. 28 Ticarcillin/clavulanic acid, the only parenteral betalactam/inhibitor combination available in Australia, has been used in such situations. In this study piperacillin/ tazobactam had better activity than ticarcillin/clavulanic acid against some Gram negative isolates. In the case of E. coli and Klebsiella species, our most common Gram negative isolates, antibiotic susceptibility patterns indicate that a small proportion of these organisms are producing TEM and SHV enzymes at high levels. The amount of TEM enzyme produced by E. coli has been found to vary 100fold among strains. ~8 As the level of enzyme activity increases, the amount of inhibitor required to restore beta-
17 1
lactam activity correspondingly increases. Livermore29 found that clavulanic acid geometric mean titres of 2.9-12.7 mg/L were required to reduce ticarcillin MICs to 16 mg/L in 36 E. coli isolates producing varying levels of TEM beta-lactamase. Corresponding tazobactam levels to reduce piperacillin MICs to 16 mg/L were 1.3-5.2 mg/L. Thus the fixed concentration of 2 mg/L for calvulanic acid was inadequate to overcome enzyme production in the hyper-producing strains. The fixed tazobactam concentration of 4 m g ~ was more successful, resulting in more strains susceptible to piperacillin/tazobactam. It is difficult to predict whether this difference would be manifest in vivo. Other differences in piperacillin/tazobactam and ticarcillin/ clavulanic acid may reflect inherently better susceptibility to piperacillin than to ticarcillin (as in the case of Pseudomonas aeruginosa). Activity of piperacillin/ tazobactam was good against all Gram positive isolates with the exception of methicillin resistant staphylococci and E. faecium. In conclusion, piperacillin/tazobactam had good activity against Bacteroides fragilis group and against a range of Gram positive and negative aerobic isolates causing infections in our hospitals. Reduced piperacillin/tazobactam activity was seen among extended spectrum beta-lactamase producing KlebsietIa species and high level producers of TEM and SHV plasmid-mediated enzymes. ACKNOWLEDGEMENTS This study was supported by a grant from Lederle Laboratories. Address for correspondence: D.D., Department of Microbiology and Infectious Diseases, South Western Area Pathology Service, Liverpool Hospital, Liverpool, NSW 2170.
References 1. Roy C, Fox A, Segura C et al. Plasmid determined beta-lactamases identified in a group of 204 ampicillin-resistant Enterobacteriaceae, J Antimicrob Chemother 1983; 12: 507-10. 2. Aymes SGB. Plasmid-mediated beta-lactamases: relative clinical importance. In: Livermore DM, editor. Beta-lactamases: current perspectives. Winchester: Theracom, 1988; 31-48. 3. Sanders CC, Sanders WE. Beta-lactam resistance in Gram negative bacteria: global trends and clinical impact. Clin Infect Dis 1992; 15: 824-39. 4. Knothe H, Shah R Krcmery V e t al. Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection 1983; 11: 315-7. 5. Semders CC, Iaconis JR Bodey GP et al. Resistance to ticarcillinpotassium clavulanate among clinical isolates of the family Enterobacteriaceae: rote of PSE-1 beta-lactamase and high levels of TEM-1 and SHV-1 and problems with false susceptibility in disk diffnsion tests. Antimicrob Agents Chemother 1988; 32: 1365-9. 6. Thomson KS, Weber DA, Sanders CC et al. Beta lactamase production in members of the family Enterobacteriacae and resistance to beta-lactam-enzyme inhibitor combinations. Antimicrob Agents Chemother 1990; 34: 622-7. 7. Wiedemann B. Genetic and biochemical basis of resistance of Enterobacteriaceae to beta-lactam antibiotics. J Antimicrob Chemotber 1986; 18 Suppl B: 31-8. 8. Sanders WE Jr, Sanders CC. Inducible beta-lactamases: clinical and epidemioIogical implications for use of newer cephalosporins. Rev Infect Dis 1988; 10: 830-8.
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9. Gutman L, Kitzis M-D, Yamabe Set al. Comparative evaluation of a new beta-lactmnase inhibitor, YTR 830, combined with different betalactam antibiotics against bacteria harbouring known beta-lactamases. Antimicrob Agents Chemother 1986; 29: 955-7. 10. Eliopoulos GM, Klimm K, Ferraro MJ et al. Comparative in vitro activity of piperacillin combined with the beta-lactamase inhibitor tazobactam (YTR 830). Diagn Microbiol Infect Dis 1989; 12: 481-8. 11. Fass RJ, Prior RB. Comparative in vitro activities of piperacillintazobactam and ticarcillin-clavulanate. Antimicrob Agents Chemother 1989; 33: 1268-74. 12. Appelbaum PC, Jacobs MR, Spangler SK et al. Comparative activity of beta-lactamase inhibitor YTR 830, clavulanate and sulbactam combined with beta-lactams against beta-lactamase-producing anaerobes. Antimicrob Agents Chemother 1986; 30: 789-91.
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13. Fac!dam RR, Collins MD. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J Clin Microbiol 1989; 27: 731-43. 14. National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susc@tibility tests for bacteria that grow aerobically M7-A3. NCCLS, Villanova, PA, 1993. 15. National Committee for Clinical Laboratory Standards: Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria Mll-A3. NCCLS, Villanova, PA, 1983. 16. Tumidge J, Lawson P, Munro R et al. A national survey of antibiotic resistance in Staphylococcus aureus in Australian hospitals. Med J Aust 1989; 150: 65-72. 17. Jorgensen JH, McElmeel ML, Maher LA, Quantitative antimicrobial susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae using the E test. J Clin Microbiol 1991; 29: 109-14. 18. Seetulsingh R Hall LMC, Livermore DM. Activity of clavulanate combinations against TEM-1 beta-lactamase producing Escherichia coli isolates obtained in 1982 and 1989. J Antimicrob Chemother 1991; 27: 749-59.
Pathology (1996), 28, May 19. Moosedeen E Keeble J, Williams JD. Induction/inhibition of chromosomal beta-lactamases by beta-lactamase inhibitors. Rev Infect Dis 1986; 8 Suppl: $562-8. 20. Klare I, Rodloff AC, Wagner J e t al. Overproduction of a penicillinbinding protein is the only mechanism of penicillin resistance in Enterococcusfaecium. Antimicrob Agents Chemother 1992; 36: 7837. 21. Tnmidge J. Foreword: Resistance in Intensive Care Units 1990. Monograph published by Merck Sharp and Dohme, 1992. 22. Wu PJ, Shannon K, Phillips I. Effect of hyper production of TEM-1 beta-lactamase on in vitro susceptibility of E. coli to beta lactam antibiotics. Antimicrob Agents Chemother 1994; 38: 494-8. 23. Livermore DM. Beta-lactamases in laboratory and clinical resistance. Clin Micro Rev 1995; 8: 557-84. 24. Rawlinson W, Sorrell TC, Munro R. Infection in neutropaenic patients. Aust NZ J Med 1988; 18: 815-7. 25. Brismar B, Malmborg AS, Tunevall G e t al. Piperacillin-tazobactam versus imipenem for treatment o:f intra-abdominal infections. Antimicrob Agents Chemother 1992; 36: 2766-77. 26. Mouton Y, Leroy O, Beuscart C et al. and Study Group. Efficacy, safety and tolerance of parenteral piperacillin/tazobactam in the treatment of patients with lower respiratory tract infections. J Antimicrob Chemother 1993; 31 Suppl A: 87-95. 27. Marie JR Vekhoff A, Cony-Makhoul P e t al. Randomised trial of piperacillin/tazobactam plus amikacin versus ceftazidime plus amikacin in 222 febrile neutropenic patients. ICAAC 1993, poster 647. 28. Kuye O, Teal J, De Vries VG et al. Safety profile of piperacillin/tazobactam in phase I and phase ItI clinical studies. J Antimicrob Chemother 1993; 31 Suppl A: 113~24. 29. Livermore DM. Determinants of the activity of beta-lactamase inhibitor combinalions. J Antimicrob Chemother 1993; 31 Suppl A: 921.
AN EVALUATION OF THE IN VITRO ACTIVITY OF PIPERACILLIN/TAZOBACTAM D. DALEY*, L. MULGRAVE'~, R. MUNRO*, S. NEVILLE*, H. SMITH~ AND
W. DIMECH$
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*Department of Microbiology and Infectious Diseases, South Western Area Pathology Service, Liverpool Hospital, Liverpool, NSW; ?Department of Microbiology; WA Centre for Pathology and Medical Research, Nedlands, WA; and ~:Department of Microbiology, Royal Melbourne Hospital, Melbourne, Vic
Summary
Key words: Piperaciltin, tazobactam, staphylococci.
Tazobactam is a new, irreversible inhibitor of bacterial betalactamases of staphylococci, piasmid-mediated beta-lactamases of the TEM and SHV types found in Escherichia coil and Klebsielta species and beta-lactamases of anerobes such as Bacteroides species. Its combination with piperacillin, a broad spectrum ureido-penicillin, would be expected to improve the activity of piperacillin against staphylococci, TEM and SHV betalactamase producing Gram negative bacteria and anerobes. Minimal inhibitory concentrations (MIC) of piperacillin/ tazobactam were determined for 1952 individual patient isolates of Gram positive and negative bacteria causing significant infections and compared with MIC values for cefotaxime, ceftazidime, ciprofloxacin, imipenem, ticarcillin/clavulanic acid. MlCs were determined by agar dilution (NCCLS 1990 and 1992). Piperacillin/tazobactam had excellent activity against methicillin susceptible staphylococci, Streptococcus pneumoniae, Haemophilus influenza& enterococci and organisms of the Bacteroides fragilis group. It was also active against the majority of Enterobacteriaceae and Pseudomonas aeruginosa isolates tested. It was not active against extended spectrum beta-lactamase (ESBL) producing Klebsiella species and some high level TEM and SHV beta-lactamase producing E. coil and Klebsiella species. Activity against Gram negative organisms capable of producing chromosomally mediated beta-lactamases was good, since in most organisms tested, the enzymes were not induced in sufficient quantities to cause antibiotic resistance. However some Enterobacter species were derepressed hyperproducing mutants; these isolates showed resistance to piperacillin/tazobactam since tazobactam does not inhibit these Class I beta tactamases. Activity was superior to ticarcillin/ clavulanic acid for Gram negative rods. lmipenem was the most active agent against ESBL producing Klebsiella species. Piperacillin/tazobactam has a suitable spectrum of activity in vitro to suggest its use in monotherapy of mixed anerobic infections, mixed respiratory infections such as aspiration pneumonia and, in combination with an aminoglycoside, it would provide Gram positive as well as Gram negative cover of febrile episodes in immunosuppressed patients.
Accepted 12 December 1995
INTRODUCTION
Beta-lactamase production is one of the most important mechanisms of antibiotic resistance in bacteria. The large number of different beta-lactamases produced by Gram positive and negative bacteria are either plasmid- or transposon-mediated, and thus capable of dissemination to other bacteria, or determined by chromosomal DNA. Among Gram negative bacteria and staphylococci, plasmid-mediated beta-lactamases are widespread and of many different types. In Gram negative bacteria the common ones are the TEM-1, TEM-2 and SHV-1 enzymes which hydrolyse ampicillin, first generation cephalosporins and other penicillins. TEM-1 and related enzymes are now widespread in enterobacteria and are present in the majority of Escherichia coti. 1-3 Their wide distribution and broad spectrum of activity have reduced the clinical usefulness of ampicillin and first generation cephalosporins and monobactams. Organisms with higher levels of enzyme production are also resistant to extended spectrum penicillins such as ticarcillin and piperacillin. Recently point mutations in TEM-1, TEM2 and SHV-1 enzymes have resulted in more extended spectrum beta-lactamases which hydrolyse third and fourth generation cephalosporins. First described in Ktebsiella species in Germany,4 these enzymes are now found worldwide especially in E. coli and KlebsielIa species. 3 In addition, hyper-production of TEM-1 and SHV-1 enzymes has resulted in increased resistance to combinations of betaIactam antibiotics with beta-lactam inhibitors) '6 The most important chromosomalty-determined betalactamases are the class 1 enzymes which occur in enterobacteria and Pseudomonas aeruginosa; they hydrolyse a wide range of cephalosporins and broad spectrum penicillins. Most Gram negative bacteria possess the chromosomal information to produce beta-lactamases but production is in insufficient quantities to cause antibiotic resistance. In Enterobacter, Citrobacter, Serratia, Proteus
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vulgaris, Morganella, Providencia species, and Pseudomonas aeruginosa the enzymes are inducible but these organisms remain susceptible to many new broad spectrum penicillins and cephalosporins since the antibiotics are poor inducers. However in all these species there are mutant subpopulations which constitutively produce class 1 enzymes at high levels] Therefore the use of beta-lactam antibiotics should be avoided in infections caused by organisms producing inducible beta-lactamases since selection of resistant mutants is possible with resultant treatment failure. ~ Piperacillin is a broad spectrum ureido-penicillin which is inactivated by staphylococcal beta-lactamases, by some of the TEM and SHV enzymes of Gram negative bacteria and, although inactivated by class 1 enzymes, retains in vittv activity against inducible enzyme producing organisms because in itself it is a poor inducer of beta-lactamases. However organisms constitutively producing class 1 enzymes are not susceptible to piperacillin. In addition, piperacillin is inactivated by" beta-lactamases of Bacteroides species. Tazobactam is a penicillanic acid sulphone which can inhibit beta-lactamases of staphylococci, some of the common plasmid-mediated TEM and SHV beta-lactamases of E. coli, H. influenzae and KIebsiella species and betalactamases of anerobes such as Bacteroides species. 922 It does not inhibit class 1 chromosomally-determined betalactamases found in Enterobacter, Citrobacter, Serratia, Proteus vulgaris, Pseudomonas, and similar species. The combination of tazobactam with piperaciltin would be expected to restore the activity of piperacillin against those organisms producing beta-lactamases inhibited by tazobactam. The present study was undertaken to investigate the efficacy of piperacillin/tazobactam in vitro against 1,952 isolates causing significant infections in Australian hospitals and to compare its activity with ceftazidime, cefotaxime, imipenem, ciprofloxacin and ticarcillin/clavulanic acid.
MATERIALS AND METHODS Organism~ A total of 1,952 clinical isolates were collected and tested in 3 Australian states. The strains represent a broad cross section of mostly hospital acquired isolates that were found to be causing significant sepsis. From New South Wales, the South Western Area Pathology Service tested 530 isolates from one tertiary referral hospital and 5 smaller hospitals with a total of 1,500 beds. The Microbiology Department of the Royal Melboume Hospital, Victoria and the 660 bed Sir Charles Gairdner Hospital, Perth, Western Australia, both ternary teaching hospitals, tested 459 and 940 strains respectively. The latter collection included 33 extended-spectrum beta-lactamase producing strains collected since they first appeared in 1989 from hospitals in all Australian mainland states. All were unique patient isolates from clinically significant infections. In particular, isolates of coagulase negative staphylococci and enterococci were only included if from normally sterile sites or documented intravascular infections, Similarly, isolates of S. pneumoniae and H. influenzae were mostly from sterile sites. Respiratory isolates were only included from definite cases of pneumonia. Identification of organisms was as follows: Gram negative rods were identified using either Vitek AlVlS or the ATB32E and ATB32GN systems (Bit Merieux SA, 69280 Marcy l'Etoile, France). Enterococci were identified using conventional biochemical media according to Facklam's criteria ~3 and S. pneumoniae on the basis of optochin susceptibility. H. influenzae was identified on the basis of X and V requirements.
TABLE 1 Organisms included in the study Organism
Escherichia coli Klebsiella spp. total ESBL 1 Klebsiella spp. Enterobacter spp. total Cittvbacter/Serratia spp. total Morganella/Pmteus/Providencia spp. total Proteus mirabilis Pseudomonas aeruginosa Acinetobacter calcoaceticus Staphylococcus aureus Coagulase negative Staphylococcus spp. Streptococcus pneumoniae Enterococcus spp. total Haemophilus influenzae Bacteroides fragitis group
Total
Number tested 528 224 44 142 84 45 104 88 40 433 28 45 109 59 23 1952
Staphylococci were identified with tube and slide tests for coagulase, DNAse and phosphatase production, anerobic fermentation of mannitol and hydrolysis with Tween 80. Anerobes were identified using the ATB32A system (Bit Merieux SA, 69280 Marcy l'Etoile, France).
Antibiotic susceptibility testing Minimal inhibitory concentrations (MIC) for all organisms except H. influenzae were determined using the agar dilution methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS)J 4'15 The only variation to that method was the incorporation of p-(4-nitrophenyt)-glycerol 43 mg/L (PNPG) into all plates except those involving specific organisms such as S. pneumoniae and anerobes. PNPG prevents swarming of Proteus species and has been previously shown not to influence MIC results. 16 Antibiotics tested were obtained from the manufacturer as pure substances of known potency. All antibiotic-containing agar plates were used within 72 hrs of preparation, with the exception of imipenem which was used on the day of preparation. Doubling dilutions were prepared for the following antibiotics: piperacillin 0.004-256 mg/L with a fixed concentration of tazobactam of 4 mg/L in each plate; cefotaxime 0.06-128 mg/L; imipenem 0.06-64 mg/L; ceftazidime 0.06-64 mg/L; ticarcillin 0.06-256 mg/L with a fixed concentration of clavulanic acid of 2 mg/L in each plate and ciprofloxacin 0.06-8 mg/L. MICs for H. influenzae isolates were determined using the E test (AB Biodisk, Pyramidvagen 7, 5-17136 Solina, Sweden)) 7
tntra-laboratory reproducibility Each laboratory tested control organisms as stipulated by the NCCLS method with each antibiotic tested. In addition every 25th organism tested at each centre was re-tested at the other centres. Organisms exchanged for re-testing were designed to occur randomly, but also to represent each group of test organisms adequately. The MIC to inhibit 50% (MIC50) and 90% (MIC90) of isolates was calculated as well as the % resistance at breakpoint concentrations recommended by the NCCLS.
RESULTS All control organisms tested gave satisfactory results according to NCCLS guidelines. A total of 28 organisms were exchanged between testing laboratories. All MIC results were within one doubling dilution of the MIC determined by the referring laboratory. These results indicated adequate intra-laboratory reproducibility and allowed for pooling of results. Table 1 summarizes the identification and numbers of organisms tested. Table 2 documents MIC50 and MIC90 determinations. Table 3 summarizes the per cent resistance at breakpoints recommended by the
AN EVALUATIONOF THE IN VITROACTIVITYOF PIPERACILLIN/TAZOBACTAM TABLE2 Minimal inhibitory concentrations (MIC) for 1,952 clinically significant isolates Organism number)
Range
MIC50
MIC90
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E. coli (528) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Ktebsiella spp. (180) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Klebsiella spp. (ESBL 44) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Enterobacter spp. (142) piperacillin/tazobactam ticarcillirdclavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Citrobacter/Serratia spp, (84) piperacitlin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin
0.25 - 128.0 0.25 ->256.0 0.06 2.0 0.03 - 16.0 0.03 8.0 0.0080.5
1.0 2.0 0.06 0.06 0.06 0.015
0.25 ->256.0 0.5 -->256.0 0.06 2.0 0.03 8,0 0.03 - 16.0 0.0081.0
1.0 1.0 0.06 0.06 0.06 0.03
1.0 ->256.0 8.0 ->256.0 0.1251.0 4.0 - >64.0 0.03 - 16.0 0.0168.0
8.0 32.0 0.125 4.0 4.0 t.0
64.0 128.0 0.25 64.0 16.0 2.0
0.004- 128.0 0.25 ->256.0 0.1254.0 0.03 - >64.0 0.03 ->128.0 0.0082.0
2.0 4.0 0.125 0.25 0.125 0.03
16.0 128.0 1.0 32.0 32.0 0.O6
0.25 - 256.0 0.5 ->256.0 0.06 8.0 0.03 - >64.0 0.03 ->128.0 0.0081.0
2.0 4.0 0.25 0.25 0.125 0.06
8.0 64.0 2.0 1.0 8.0 0.12
2.0 8.0 2.0 16.0 0.5 0.5
2.0 32.0 0.25 0.25 0.25 0.03 4.0 4.0 0.25 0.25 0.125 0.06
169
TABLE 2 Continued Organism (number) Coagulase negative Staphylococcal (28) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin
Range
MIC50
0.125- 256.0 0.125->256.0 0 . 0 6 - 64.0 4.0 - >64.0 0.25 ->128.0 0.1250.5
0.5 1.0 0.06 8.0 2,0 0,25
0.004- 0.25 0.06 4.0 0,06 0.06 2.0 0.06 0.25 2.0
0.001 0.25 -<0,06 0,12: -<0.06 1.0
MIC90
16.0 64.0 8.0 >64.0 >128.0 0.25
Streptococcus pneumoniae (45) piperacillin/tazobactam ticarciUin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Enterococci (109) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Haemophilus influenzae (59) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Bacteroidesfragilis gp (23) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin
0.06 ->256.0 1.0 ->256.0 0 . 0 6 - 64.0 0.06 - >64.0 0.03 ->128.0 0.125- >8.0
2.0 32,0 1.0 >64.0 64.0 1.0
<0.016-0.25 0.0471.5 0.064-- >32.0 0.0471.5 0.003 -0.047 0.003- 0.19
<0,01~ 0.12: 0.75
0.004->256.0 0.06 - 256.0 0.06 - 16.0 16.0 - >64.0 32.0 ->128.0 1.0 - > 8 . 0
0.5 4,0 0.25 64.0 64.0 4.0
0,12:
0.0E 0.01:
0.015 0.5 -<0.06 0.25 -<0.06 2.0 4.0 64.0 2.0 >64.0 >128.0 2.0 0.094 0.5 >32.0 0.25 0.032 0.016 4.0 32.0 2.0 >64.0 >128.0 >8.0
Morganella/Proteus/ Providencia spp, (45) piperacillirdtazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Proteus mirabilis (104) piperacillin/tazobactam ticarciUin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Pseudomonas aeruginosa (88) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Acinetobacter calcoaceticus (40) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin Staphylococcus aureus (433) piperacillin/tazobactam ticarcillin/clavulanic acid imipenem ceftazidime cefotaxime ciprofloxacin
0.1250.250.06 0.060.03 0.008-
4.0 32.0 2.0 32.0 8.0 8.0
0,25 1.0 1.0
0.1250.25 0.1250.03 0.030.008-
2.0 16.0 4.0 0.25 0.25 0.5
0.25 0.5 2.0 0.06 0.06 0.03
0.125
0.06 0.015
0.5 1.0 4.0 0.06 0.06 0.06
0.5 ->256.0 2.0 ->256.0 0.5 - 32.0 0.5 - 16.0 2.0 ->128,0 0 . 0 6 - >8.0
4.0 16.0 2.0 2.0 32.0 0.125
32.0 64.0 16.0 8.0 128.0 1.0
0.25 - 64.0 0.5 - 128.0 0.1251.0 0.03 - 16.0 0.125- 64.0 0.0158,0
16.0 32.0 0.25 4.0 32.0 0.5
32.0 64.0 0.5 16.0 64.0 2.0
0.125-256.0 0.25 ->256.0 0 . 0 6 - 64.0 0.5 - >64,0 0.125->128.0 0.125- >8.0
1.0 1.0 0.125 8.0 2.0 0.5
4.0 32.0 1.0 32.0 16.0 1.0
NCCLS. Breakpoints are not provided by the NCCLS for S.
pneumoniae and enterococci for any of the antibiotics tested, for ticarcillin/clavulanic acid against any Gram positive organisms and for piperacillin/tazobactam and ticarcillin/clavulanic acid against H. influenzae. Activity against Gram negative organisms A g a i n s t E. coli and Klebsiella s p e c i e s , the t w o m o s t c o m m o n G r a m n e g a t i v e o r g a n i s m g r o u p s , all antibiotics t e s t e d h a d g o o d activity w i t h l o w levels o f r e s i s t a n c e e x c e p t a m o n g E S B L p r o d u c i n g KlebsielIa s p e c i e s . T h e s e o r g a n i s m s s h o w e d characteristically h i g h levels o f resistance to b e t a - l a c t a m s w i t h the e x c e p t i o n o f i m i p e n e m . N e i t h e r t a z o b a c t a m n o r clavulanic acid i n h i b i t e d t h e s e E S B L s and the o r g a n i s m s w e r e r e s i s t a n t to b o t h p i p e r a c i l l i n / t a z o b a c t a m and ticarcillin/clavulanic acid. N i n e t e e n p e r c e n t o f o t h e r w i s e s u s c e p t i b l e E. coli and 7 % o f n o n - E S B L Klebsiella s p e c i e s w e r e r e s i s t a n t to ticarcillin/ clavulanic acid b u t o n l y 4 % o f Klebsiella s p e c i e s and 1% o f E. coIi w e r e also r e s i s t a n t to p i p e r a c i l l i n / t a z o b a c t a m . T h e s e o r g a n i s m s s h o w e d o n l y l o w levels o f r e s i s t a n c e to o t h e r c e p h a l o s p o r i n s tested, T h e antibiotic s u s c e p t i b i l i t y p a t t e r n s o f t h e s e E. coli and Klebsiella s p e c i e s are c h a r a c t e r i s t i c o f o r g a n i s m s w i t h h i g h levels o f p r o d u c t i o n o f T E M o r S H V b e t a - l a c t a m a s e s 18 and e x h i b i t i n g r e s i s t a n c e to ampicillin, a m o x y c i l l i n / c l a v u l a n i c acid, t i c a r c i l l i n a n d p i p e r a c i l l i n (results not s h o w n ) . T h e d i f f e r e n c e in the f r e q u e n c y o f
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TABLE3 Per cent resistance at NCCLS breakpoints Organism
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(Number) E. coli (528) Klebsiella spp. (180) KtebsielIa spp. (ESBL44) Enterobacter spp. (142) Citrobacter/Serratia spp. (84) Morganetla/Providencia/ Proteus spp. (45) P. mirabilis (104) Ps. aeruginosa (88) Acinetobacter calcoaceticus (40) S. aureus (433) Coagulase neg. Staphylococcal(28) Haemophilus influenzae (59) Bacteroidesfragilis gp (23) Breakpoints mg/L) Staphylococci Gram neg. rods Pseudomonas Anerobes
Antibiotic Ceftax
Ceftaz
Imipen
Pip/Taz
Tic/Clay
Cipro
0% 2% 16% 18% 7%
1% 0% 27% 19% 10%
0% 0% 0% 0% 1%
1% 4% 13% 11% 4%
19% 7% 70% 28% 17%
0% 0% 11% 1% 0%
0% 0% 71% 80% 11% 25% 0% 74%
11% 0% 6% 12% 18% 37% 0%
0% 0% 29% 0% 8% 14% 19% 4%
0% 0% 2% 50% 9% 12%
2% 0% 7% 60%
4%
4%
11% 0% 10% 20% 4% 0% 0%
8 8 8
8 8 8
32
8
16 64 ~32/4
16 64 128
1 1 1
Ceftax=Cefotaxime Ceftaz=Ceftazidime Imipen=Irnipenem Pip/Taz=Piperacillin/Tazobactam Tic/Clav=Ticarcillin/Clavulanicacid Cipro=Ciprofloxacin resistance between the two beta-lactam/beta-lactamase inhibitors probably reflects differing amounts of betalactamase produced and the different concentrations of inhibitor in the plates (2 mg/L of clavulanic acid versus 4 mg/L oftazobactam). The divergence in MIC50 and MIC90 among E. coli for ticarcillin/clavulanic acid (MIC50 2 rag/L, MIC90 32 rag/L) suggests a subpopulation of high level enzyme producers. Isolates of Pseudomonas aeruginosa were among the most antibiotic resistant. Resistance to imipenem, cefotaxime, ticarcillin/clavulanic acid and piperacillin/tazobactam may be due to production of the class l a m p C beta-lactamase which is not inhibited by clavulanic acid or tazobactam along with a porin mutation specific for imipenem. All Proteus mirabilis strains examined were uniformly susceptible to all six agents tested. Some strains of Enterobacter, Citrobacter and Serratia species harboured derepressed chromosomal beta-lactamases, rendering them resistant to all the beta-lactams tested including both inhibitor combinations. Pooled results showed the MICs of piperacillin/tazobactam for these isolates to be consistently lower than that for ticarcillin/clavulanic acid but only by a single doubling dilution. When the results were analysed by Australian state, two of three states recorded a four-fold difference favouring piperacillin/tazobactam. The difference in susceptibility between piperacillin/tazobactam and ticarcillin/clavulanic acid probably results from the different amounts of each inhibitor used in testing. Alternatively clavulanic acid (but not tazobactam), which is a strong inducer of class I enzymes, 19 may have facilitated destruction of ticarcillin by in vitro enzyme induction. Activity against staphylococci Data presented in the tables include both MRSA and MSSA, as the data were not analysed separately.
Piperacillin/tazobactam had very good activity against methicillin sensitive S. aureus isolates. Low levels of resistance were also seen for other antibiotics tested against these strains. All resistance to beta-lactam antibiotics seen in staphylococci occurred in methicillin resistant isolates. It is generally considered that in vitro susceptibility results for beta-lactam antibiotics are inaccurate for MRSA isolates and should be regarded as resistant to all beta-lactam antibiotics. Ciprofloxacin resistance was common among MRSA isolates. Susceptibility of coagulase negative staphylococci to beta-lactams including piperacillin/ tazobactam was generally the same as susceptibility to methicillin. Activity against S. pneumoniae and enterococci
Very low MICs were seen in all antibiotics tested against these organisms. There were no penicillin-resistant isolates of S. pneumoniae in the collections tested. A small number of enterococcal isolates were E. faecium - these were resistant to all beta-lactams tested including piperacillin/tazobactam. Broad-spectrum beta-lactam resistance in these organisms is due to mutation of penicillin-binding proteins. 2° Activity against H. influenzae
With the exception of imipenem (19% resistant), all strains tested were found to be susceptible to the remaining antibiotics tested. Activity against Bacteroides fragilis group High MICs were seen for the third generation cephalosporins tested and to ciprofloxacin. Piperacillin/tazobactam had good activity against these organisms. NCCLS breakpoints for anerobes were not available for ceftazidime and ciprofloxacin.
AN EVALUATIONOF THE IN VITRO ACT[VITY OF PIPERACILLIN/TAZOBACTAM
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DISCUSSION Antibiotic susceptibility patterns will vary in different hospitals reflecting the distribution of organisms causing infections and their resistance mechanisms. Piperacillin/ tazobactam has the potential to provide broad-spectrum activity against staphylococci, streptococci, aerobic Gram negative rods and anerobes which are resistant to piperacillin by virtue of beta-lactamase production if the enzyme can be inhibited by tazobactam, In this study piperacillin/tazobactam and imipenem were the most active antibiotics tested. While imipenem retained activity aginst ESBL producing Klebsiella species, lower levels of resistance were seen to piperacillin/tazobactam than to imipenem among Ps. aeruginosa. ESBL producing Klebsiella pneumoniae and strains of Enterobacteriaceae that hyperproduce TEM and SHV beta-lactamases are an increasing problem in Australian hospitals. ~1Although ESBL enzymes are known to be susceptible to inhibition by clavulanic acid and tazobactam, the production of these enzymes in addition to the SHV-1 and TEM-1 enzymes is tantamount to hyperproduction. HyperTEM-1 production in E. coli is a mechanism proven to overwhelm clavulanic acid at clinically achievable levels.22 This mechanism is the probable explanation for the poor activity of inhibitor combinations toward ESBL strains shown by the pooled results (MIC90 range 64-128 mg/L). A second possible explanation is the occurrence of mutants with reduced affinity of their betalactamases to the inhibitors. However such variants are rare at the present time, although this situation may change in the future with greater clinical use of inhibitor combinations.23 Because of its broad antibacterial effects, piperacillin/ tazobactam has potential as monotherapy in mixed infections and also as part of empirical therapy in immunosuppressed patients where activity against staphylococci is as necessary as Gram negative cover.24 Unless methicillin resistance is common in staphylococcal isolates piperacillin/ t~obactam can provide both Gram positive and Gram negative cover in conjunction with an aminoglycoside in these circumstances. Piperacillin/tazobactam activity against S. pneumoniae and H. influenzae also make it attractive as monotherapy for mixed respiratory infections such as aspiration pneumonia. Other mixed anerobic infections in the gastrointestinal and genitourinary systems are similar targets for piperacillin/tazobactam monotherapy. Reports of clinical trials with piperacillin/tazobactam indicate its usefulness in intra-abdominal infections, 25lower respiratory tract infection, 26 and febrile neutropenic patientsY The safety and efficacy profile of piperacillin/ tazobactam in over 1,200 patients was excellent. 28 Ticarcillin/clavulanic acid, the only parenteral betalactam/inhibitor combination available in Australia, has been used in such situations. In this study piperacillin/ tazobactam had better activity than ticarcillin/clavulanic acid against some Gram negative isolates. In the case of E. coli and Klebsiella species, our most common Gram negative isolates, antibiotic susceptibility patterns indicate that a small proportion of these organisms are producing TEM and SHV enzymes at high levels. The amount of TEM enzyme produced by E. coli has been found to vary 100fold among strains. ~8 As the level of enzyme activity increases, the amount of inhibitor required to restore beta-
17 1
lactam activity correspondingly increases. Livermore29 found that clavulanic acid geometric mean titres of 2.9-12.7 mg/L were required to reduce ticarcillin MICs to 16 mg/L in 36 E. coli isolates producing varying levels of TEM beta-lactamase. Corresponding tazobactam levels to reduce piperacillin MICs to 16 mg/L were 1.3-5.2 mg/L. Thus the fixed concentration of 2 mg/L for calvulanic acid was inadequate to overcome enzyme production in the hyper-producing strains. The fixed tazobactam concentration of 4 m g ~ was more successful, resulting in more strains susceptible to piperacillin/tazobactam. It is difficult to predict whether this difference would be manifest in vivo. Other differences in piperacillin/tazobactam and ticarcillin/ clavulanic acid may reflect inherently better susceptibility to piperacillin than to ticarcillin (as in the case of Pseudomonas aeruginosa). Activity of piperacillin/ tazobactam was good against all Gram positive isolates with the exception of methicillin resistant staphylococci and E. faecium. In conclusion, piperacillin/tazobactam had good activity against Bacteroides fragilis group and against a range of Gram positive and negative aerobic isolates causing infections in our hospitals. Reduced piperacillin/tazobactam activity was seen among extended spectrum beta-lactamase producing KlebsietIa species and high level producers of TEM and SHV plasmid-mediated enzymes. ACKNOWLEDGEMENTS This study was supported by a grant from Lederle Laboratories. Address for correspondence: D.D., Department of Microbiology and Infectious Diseases, South Western Area Pathology Service, Liverpool Hospital, Liverpool, NSW 2170.
References 1. Roy C, Fox A, Segura C et al. Plasmid determined beta-lactamases identified in a group of 204 ampicillin-resistant Enterobacteriaceae, J Antimicrob Chemother 1983; 12: 507-10. 2. Aymes SGB. Plasmid-mediated beta-lactamases: relative clinical importance. In: Livermore DM, editor. Beta-lactamases: current perspectives. Winchester: Theracom, 1988; 31-48. 3. Sanders CC, Sanders WE. Beta-lactam resistance in Gram negative bacteria: global trends and clinical impact. Clin Infect Dis 1992; 15: 824-39. 4. Knothe H, Shah R Krcmery V e t al. Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection 1983; 11: 315-7. 5. Semders CC, Iaconis JR Bodey GP et al. Resistance to ticarcillinpotassium clavulanate among clinical isolates of the family Enterobacteriaceae: rote of PSE-1 beta-lactamase and high levels of TEM-1 and SHV-1 and problems with false susceptibility in disk diffnsion tests. Antimicrob Agents Chemother 1988; 32: 1365-9. 6. Thomson KS, Weber DA, Sanders CC et al. Beta lactamase production in members of the family Enterobacteriacae and resistance to beta-lactam-enzyme inhibitor combinations. Antimicrob Agents Chemother 1990; 34: 622-7. 7. Wiedemann B. Genetic and biochemical basis of resistance of Enterobacteriaceae to beta-lactam antibiotics. J Antimicrob Chemotber 1986; 18 Suppl B: 31-8. 8. Sanders WE Jr, Sanders CC. Inducible beta-lactamases: clinical and epidemioIogical implications for use of newer cephalosporins. Rev Infect Dis 1988; 10: 830-8.
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