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1 ORIGINAL ARTICLE /j x Comparative assessment of inoculum effects on the antimicrobial activity of amoxycillin clavulanate and piperacillin tazobactam with extended-spectrum b-lactamase-producing and extended-spectrum b-lactamase-non-producing Escherichia coli isolates. L. López-Cerero 1, E. Picón 2, C. Morillo 2, J. R. Hernández 2, F. Docobo 3, J. Pachón 3, J. Rodríguez-Baño 4 and A. Pascual 5 1) Department of Microbiology, University Hospital Virgen Macarena, 2) Department of Microbiology, Faculty of Medicine, University of Seville, 3) Infectious Diseases Unit, University Hospital Virgen del Rocío, 4) Infectious Diseases Unit, University Hospital Virgen Macarena and 5) Department of Microbiology, University of Seville and University Hospital Virgen Macarena, Seville, Spain Abstract A significant inoculum-size effect has been observed with piperacillin tazobactam, and has been associated with b-lactamase production in extended-spectrum b-lactamase (ESBL) producers. This association has not been previously studied in the case of amoxycillin clavulanate. Piperacillin tazobactam and amoxycillin clavulanate were compared, using high inocula of susceptible strains either harbouring ESBLs or not. Two non-esbl-producing and 15 amoxycillin clavulanate-susceptible and piperacillin tazobactam-susceptible ESBL-producing Escherichia coli isolates, and their respective transconjugants, were tested in dilution susceptibility tests using standard and 100-fold higher inocula. Three ESBL-producing strains and E. coli ATCC were selected for time-kill studies using standard and high initial inocula. At high inocula, MICs of piperacillin increased >eight-fold for non-esbl-producing strains, and MICs of piperacillin tazobactam (8 : 1 ratio or with tazobactam fixed at 4 mg/l) increased>eight-fold for all ESBL-producing strains. However, amoxycillin MICs were not affected by a high inoculum with non-esbl-producing strains, whereas the MICs of amoxycillin clavulanate (2 : 1 and 4 : 1) increased four-fold for ESBL producers, using the broth and agar dilution methods. In kinetic studies at a high inoculum, amoxycillin and amoxycillin clavulanate were bactericidal against E. coli ATCC 25922, whereas piperacillin and piperacillin tazobactam yielded decreases of <1 log 10 CFU/mL. Similarly, at a high inoculum, only amoxycillin clavulanate was able to maintain bactericidal rates of killing over 24 h against the ESBL-positive E. coli isolates. The stability of amoxycillin clavulanate and the contrasting results obtained with piperacillin tazobactam against high inocula of ESBL-non-producing and ESBL-producing E. coli strains appear to be related to aspects other than the amount of b-lactamase production. Keywords: Amoxycillin clavulanate, extended-spectrum b-lactamase, inoculum effect, piperacillin tazobactam Original Submission: 12 December 2008; Revised Submission: 15 April 2009; Accepted: 19 April 2009 Editor: R. Canton Article published online: 15 July 2009 Clin Microbiol Infect 2010; 16: Corresponding author and reprint requests: L. López-Cerero, Departamento de Microbiología. Hospital Universitario Virgen Macarena, Avda. Dr Fedriani s/n Sevilla, Spain llopez@us.es Introduction Extended-spectrum b-lactamase (ESBL) production is considered to be one of the most important mechanisms of resistance against b-lactams, except for carbapenems, in Escherichia coli and Klebsiella species [1,2]. Concomitant resistance to other antibiotics, such as fluoroquinolones [3], reduces the treatment options. ESBL-producing organisms have frequently appeared to be susceptible to piperacillin tazobactam in vitro [4,5], and several authors have suggested that this combination could be used as therapy [6,7]. However, the in vitro activity of piperacillin tazobactam has been called into question because of a substantial inoculum-size effect observed in dilution susceptibility tests using ESBL producers [8 11]. Although the available data on amoxycillin clavulanate susceptibility of ESBL-producing strains are scarce, in some areas this combination has been shown to be active in vitro against 69% [12] and 85% [5] of ESBL-positive Enterobacteria- Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases

2 CMI López-Cerero et al. Inoculum effect on antibiotic activity 133 ceae isolates. Frequently, the recommendations concerning the utility of b-lactams associated with b-lactamase inhibitors are based on piperacillin tazobatam data, but studies exploring the effect of a large inoculum on amoxycillin clavulanate activity with ESBL producers have not been carried out. The present study, using susceptibility tests and a timekill methodology, examines whether both combinations, piperacillin tazobactam and amoxycillin clavulanate, behave similarly against large inocula of non-esbl-producing and ESBL-producing E. coli isolates. Materials and Methods ATCC was included as a control. The studies were performed at 2 MIC, 4 MIC and 8 MIC, with initial log-phase growth inocula of (standard inoculum) [14] and CFU/mL. Amoxycillin clavulanate was added at a 2 : 1 ratio, and piperacillin tazobactam was used with a fixed 4 mg/l concentration of tazobactam. Samples were removed after 2, 4, 6, 8 and 24 h of incubation at 37 C and plated using an automatic spiral spreading instrument (IUL, Barcelona, Spain) for viable colony counts. The lower limit of detection was set at log CFU/mL. Antibiotic carryover was evaluated according to CLSI guidelines [14]. MICs of piperacillin tazobactam were determined for regrowth organisms. Strains Tests were performed with 15 clonally unrelated piperacillin tazobactam-susceptible (MIC 90 2 mg/l) and amoxycillin clavulanate-susceptible (MIC 90 4 mg/l) ESBL-producing E. coli strains [12] that expressed sequence-confirmed SHV (three SHV-12 and two SHV-2), TEM (one TEM-3, one TEM-4, one TEM-10, one TEM-52, and one TEM-116) and CTX-M (three CTX- M-14, one CTX-M-9, and one CTX-M-10) enzymes. Their 15 respective transconjugants, mated to E. coli J-53 Az R (resistant to sodium azide) by conjunction [12], were also studied. Non- ESBL-producing strains, E. coli ATCC and E. coli J-53 Az R, were included as controls. Antimicrobial susceptibility testing Antibiotic susceptibilities were determined using the broth microdilution and agar dilution methods in Mueller Hinton broth and Mueller Hinton agar (Oxoid, Cambridge, UK), respectively [13]. Two different inocula were employed whose densities differed 100-fold: (standard inoculum) [13] and CFU/mL for the broth method, and (standard inoculum) [13] and CFU/spot for the agar method. Tests were carried out in duplicate, and inoculumsize controls were provided by quantitative subculture. Antibiotics were obtained from the following sources: amoxycillin, piperacillin and tazobactam from Sigma-Aldrich (Madrid, Spain) and clavulanate from GlaxoSmithKline (Brentford, UK). The b-lactam b-lactamase inhibitor combinations were tested as follows: amoxycillin clavulanate at 2 : 1 and 4 : 1, and piperacillin tazobactam at 8 : 1 with a fixed concentration of 4 mg/l tazobactam. An inoculum effect was defined as an eight-fold or greater increase in MIC value upon testing with the highest inoculum [11]. Time-kill assays Three clinical strains producing each type of ESBL were selected for production of the time-kill curves, and E. coli Results Studies with non-esbl-producing E. coli isolates In the case of the non-esbl-producing strains, the MIC values of amoxycillin or the two amoxycillin clavulanate combinations were shown not to be affected by inoculum size (Table 1). Piperacillin, however, alone or with tazobactam, whether at a fixed 4 mg/l concentration or at the ratio of 8 : 1, was associated with a pronounced inoculum effect both in broth and in agar. For E. coli ATCC at the standard inoculum, amoxycillin and piperacillin, as well as amoxycillin clavulanate and piperacillin tazobactam, were able to achieve 99.9% killing at 8 h in time-kill studies and to maintain this over 24 h at 4 MIC. With this inoculum, it appears that the TABLE 1. MICs of b-lactams and b-lactam b-lactamase inhibitors for non-extended-spectrum b-lactamase-producing Escherichia coli strains determined with two bacterial inocula Antibiotic Method MIC (mg/l) ATCC J53 Az R S H S H Amoxycillin Broth Agar Amoxycillin clavulanate (2 : 1) Broth Agar Amoxicyllin clavulanate (4 : 1) Broth Agar Piperacillin Broth 2 > Agar Piperacillin tazobactam (4 mg/l) Broth Agar Piperacillin tazobactam (8 : 1) Broth 2 > Agar S, standard inoculum for every method according to CLSI guidelines; H, 100-fold standard inoculum.

3 134 Clinical Microbiology and Infection, Volume 16 Number 2, February 2010 CMI two inhibitor combinations are very similar. Additionally, when the inoculum was increased 100-fold, amoxycillin and amoxycillin clavulanate achieved 99.9% killing at 8 h and 24 h, whereas piperacillin and piperacillin tazobactam showed decreases of <1 log 10 CFU/mL (Fig. 1). Studies with ESBL-producing E. coli Table 2 compares the variations in the MIC values of the two b-lactam b-lactamase inhibitors when the bacterial load of ESBL producers was increased 100-fold. At a high inoculum, a 8-fold increase in MIC was found for piperacillin tazobactam in broth (mean of 87-fold at the 8 : 1 ratio and 221-fold at 4 mg/l of tazobactam) and in agar (mean of 17-fold (8 : 1) and 168-fold (4 mg/l tazobactam)) with all the strains. When the inoculum was increased, the MICs of piperacillin tazobactam were 256 mg/l for 90% (at the 8 : 1 ratio) and 97% (at 4 mg/l of tazobactam) of the strains. On the other hand, neither combination of amoxycillin clavulanate appeared to be affected by a 100-fold inoculum increase, the MICs increasing <8-fold in all cases with the broth and agar methods. With the highest inoculum, six (20%) and seven (23%) strains achieved MIC values of 16 mg/l for amoxycillin clavulanate 2 : 1, and 15 (50%) and 16 (53%) strains for amoxycillin clavulanate 4 : 1, with the FIG. 1. Time-kill curves of E. coli ATCC at high inoculum for AMC (a) and PTZ (b); and time-kill curves of ESBL-producers at standard inoculum with 8 AMC MIC (c) and 8 PTZ MIC (d), and at high inoculum with 8 AMC MIC (e) and 8 PTZ MIC (f). Filled diamonds, CTX-M-14-producing strain; open squares, TEM-3-producing strain; open circles, SHV-12-producing strain; double cross, 2 MIC; cross, 4 MIC; cross, 8 MIC; broken line, lower limit of detection. TABLE 2. MICs of b-lactam b-lactamase inhibitors for both clinical and transconjugant extended-spectrum b-lactamase-producing Escherichia coli strains determined with two bacterial inocula MIC (mg/l) of antimicrobial combinations Amoxycillin clavulanate Piperacillin tazobactam 2 : 1 4 : 1 8 : 1 Tazobactam 4 mg/l Type of b-lactamase S H S H S H S H CTX-M group fi SHV group fi TEM group fi S, standard inoculum for every method according to CLSI guidelines; H, 100-fold standard inoculum.

4 CMI López-Cerero et al. Inoculum effect on antibiotic activity 135 broth and agar methods, respectively. For no strains were the MICs of amoxycillin clavulanate 32 mg/l. No differences were observed between clinical and transconjugant strains. Figure 1 shows the killing curves obtained with the three selected ESBL-producing isolates at standard and high inocula at antibiotic concentrations of 8 MIC. Bactericidal results remained similar at 2 MIC, 4 MIC, and 8 MIC. In standard conditions and at a high inoculum, amoxycillin clavulanate achieved 99.9% killing against the three ESBL producers at 8 h, and was able to maintain this over 24 h. The only difference noted was with the TEM-3-producing isolate with 2 MIC at 8 h, when amoxycillin clavulanate achieved only 99% killing at a high inoculum. However, piperacillin tazobactam achieved maximal killing against the three ESBL producers at 8 h, although the reduction in CFU/mL was <3 log 10 at high inoculum and regrowth occurred. No increased piperacillin tazobactam MIC values were observed in these cases. Discussion To the best of our knowledge, our results provide the first evidence for stability of amoxycillin clavulanate MIC values under an increased bacterial load of E. coli, regardless of ESBL production or enzyme type. Additionally, we found that a high inoculum size influences piperacillin tazobactam and amoxycillin clavulanate differently in both susceptibility tests and time-kill experiments with E. coli strains. The stability of amoxycillin clavulanate and the effect of inoculum size on piperacillin tazobactam described in this study may only apply to E. coli. It would be interesting to investigate possible differences in other enterobacteria. The effects of inoculum size on piperacillin activity have previously been investigated for non-esbl-producing strains using in vitro susceptibility tests [15] and for piperacilin tazobactam using a time-kill methodology [16]. Later studies also noted that a large inoculum had an effect on piperacillin tazobactam activity with ESBL-harbouring strains. Increases in the MIC of piperacillin tazobactam to 256 fi 512 mg/l have been observed with TEM-producing strains [9,11] and stains producing SHV-derived enzymes [11]. In our study, the impact of the bacterial load affected the three ESBL types analysed. These data agree with those of Segatore [10], who observed a significant inoculum-size effect with piperacillin tazobactam in 90% of 103 isolates producing complex b-lactamase patterns when CFU/mL was used. The presence of an inoculum effect with piperacillin tazobactam has been associated with the production of large amounts of hydrolysing b-lactamases derived from higher inocula [17]. However, this does not explain amoxycillin clavulanate stability under the same conditions, or the results obtained with non-producing strains. A study limitation was the undefined genetic background of the isolates that were employed. The promoter sequences and the number of copies of the bla genes were not available, and these aspects may modify the amount of enzyme. Nonetheless, b-lactamase-producing strains behave similarly to the ATCC E. coli isolate, which lacks any enzymatic resistance determinant, and therefore it is reasonable to assume that the amount of b-lactamase, derived from a high inoculum or from a high level of production, is not related to the inoculum effect. A potential explanation for the differences found in vitro between piperacillin tazobactam and amoxycillin clavulanate, as well as between amoxycillin and piperacillin, may depend mainly on differences in the target protein. Amoxycillin has the strongest affinity for penicillin-binding protein (PBP) 1A and PBP 2 [18], whereas piperacillin preferentially binds PBP 2 and PBP 3 [19]. There is a marked decrease in PBP 3 in stationary-phase cells, because an inverse growth-dependent mechanism directs the expression of this protein (reviewed in [20]). According to our results, when large inocula were used in the time-kill experiments, those strains that had been incubated without an antibiotic reached the stationary phase at 4 h. Piperacillin achieved 99.9% killing with a standard inoculum at 6 h, and amoxycillin at 4 h. In the in vitro conditions used in our study, large inocula may overwhelm piperacillin, allowing growth and decreased PBP 3 production before the inhibitory activity of the antibiotic becomes significant. The enhanced activity of amoxycillin clavulanate may also be related to the modest binding of clavulanate to PBP 2 [21]. Thus, there is an added effect on the E. coli PBPs that is not provided by the combination with tazobactam, an inhibitor that does not bind to E. coli PBPs. The most important question about differences observed in both b-lactam b-lactamase inhibitor combinations, when exposed to high inocula, is whether this in vitro phenomenon may, in clinical practice, have implications for treatment. Clinical failure has been observed for piperacillin tazobactam in infections caused by ESBL producers [22 24]. It would be interesting to know whether clinical failure may occur in serious infections with non-esbl-producing E. coli isolates. One way to ascertain whether in vitro differences have any clinical significance is through the use of an animal infection model. In conclusion, amoxycillin clavulanate and piperacillin tazobactam behave differently against the challenge of a high bacterial load with ESBL-producing and ESBL-non-producing E. coli isolates. Whereas amoxycillin clavulanate maintained

5 136 Clinical Microbiology and Infection, Volume 16 Number 2, February 2010 CMI its activity, piperacillin tazobactam showed a pronounced inoculum effect. Our data suggest that this inoculum effect is not related to b-lactamase production, and further investigations are needed to find other factors affecting the bactericidal activity related to high inocula and their clinical consequences. Acknowledgements This study was partially supported by REIPI (Spanish Network for Research in Infectious Diseases), Instituto de Salud Carlos III, Ministerio de Salud y Consumo (C03/14), FIS PI051019, and by a grant (75/04) from the Consejería de Salud, Junta de Andalucía, Spain. Transparency Declaration All authors declare no conflicts of interest. References 1. Pitout JD, Laupland KB. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008; 8: Cantón R, Novais A, Valverde A et al. Prevalence and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Europe. Clin Microbiol Infect 2008; 14 (suppl 1): Tolun V, Kucukbasmaci O, Torumkuney-Akbulut D et al. Relationship between ciprofloxacin resistance and extended-spectrum beta-lactamase production in Escherichia coli and Klebsiella pneumoniae strains. Clin Microbiol Infect 2004; 10: Pagani L, Migliavacca R, Luzzaro F et al. Comparative activity of piperacillin/tazobactam against clinical isolates of extended-spectrum beta-lactamase-producing Enterobacteriaceae. Chemotherapy 1998; 44: Spanu T, Luzzaro F, Perilli M et al. Occurrence of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae in Italy: implications for resistance to beta-lactams and other antimicrobial drugs. Antimicrob Agents Chemother 2002; 46: Gavin PJ, Suseno MT, Thomson RB Jr et al. Clinical correlation of the CLSI susceptibility breakpoint for piperacillin tazobactam against extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella species. Antimicrob Agents Chemother 2006; 50: Peterson LR. Antibiotic policy and prescribing strategies for therapy of extended-spectrum beta-lactamase-producing Enterobacteriaceae: the role of piperacillin tazobactam. Clin Microbiol Infect 2008; 14 (suppl 1): Jett DD, Ritchie DJ, Reichley R et al. In vitro activities of various betalactam antimicrobial agents against clinical isolates of Escherichia coli and Klebsiella spp. resistant to oxyimino cephalosporins. Antimicrob Agents Chemother 1995; 39: Leleu G, Kitzis MD, Vallois JM et al. Different ratios of the piperacillin tazobactam combinations for treatment of experimental meningitis due to Klebsiella pneumoniae producing the TEM-3 extended-spectrum beta-lactamase. Antimicrob Agents Chemother 1994; 38: Segatore B, Setacci D, Perilli M et al. Antimicrobial susceptibility of clinical isolates of Enterobacteriaceae producing complex beta-lactamase patterns including extended-spectrum enzymes. Int J Antimicrob Agents 2004; 23: Thomson KS, Moland ES. Cefepime, piperacillin tazobactam, and the inoculum effect in tests with extended-spectrum beta-lactamase-producing Enterobacteriaceae. Antimicrob Agents Chemother 2001; 45: Hernández JR, Martínez-Martínez L, Cantón R et al. Nationwide study of Escherichia coli and Klebsiella pneumoniae producing extendedspectrum beta-lactamases. Antimicrob Agents Chemother 2005; 49: Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A7. Wayne, PA: CLSI, National Committee for Clinical Laboratory Standards. Methods for determining bactericidal activity of antimicrobial agents. Approved standard M26-A. Wayne, PA: NCCLS, Soriano F, García-Corbeira P, Ponte C et al. Correlation of pharmacodynamic parameters of five beta-lactam antibiotics with therapeutic efficacies in an animal model. Antimicrob Agents Chemother 1996; 40: Goldstein EJ, Citron DM, Cherubin CE. Comparison of the inoculum effects of members of the family Enterobacteriaceae on cefoxitin and other cephalosporins, beta-lactamase inhibitor combinations, and the penicillin-derived components of these combinations. Antimicrob Agents Chemother 1991; 35: Burgess DS, Hall RG 2nd. In vitro killing of parenteral beta-lactams against standard and high inocula of extended-spectrum beta-lactamases and non-esbl producing Klebsiella pneumoniae. Diagn Microbiol Infect Dis 2004; 49: Curtis NA, Orr D, Ross GW et al. Affinities of penicillins and cephalosporins for the penicillin-binding proteins of Escherichia coli K-12 and their antibacterial activity. Antimicrob Agents Chemother 1979; 16: Mitsuyama J, Takahata M, Yasuda T et al. The mechanism of action of piperacillin-analogues in vitro; effect of the carbon number at the N-4 position of 2,3-dioxopiperazine on the outer membrane permeability, stability to beta-lactamase and binding affinity to penicillin-binding proteins. J Antibiot (Tokyo) 1987; 40: Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P. The penicillinbinding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev 2008; 32: Spratt BG, Jobanputra V, Zimmermann W. Binding of thienamycin and clavulanic acid to the penicillin-binding proteins of Escherichia coli K-12. Antimicrob Agents Chemother 1977; 12: Burgess DS, Hall RG 2nd, Lewis JS 2nd et al. Clinical and microbiologic analysis of a hospital s extended-spectrum beta-lactamase-producing isolates over a 2-year period. Pharmacotherapy 2003; 23: Paterson DL, Singh N, Gayowski T et al. Fatal infection due to extended-spectrum beta-lactamase-producing Escherichia coli: implications for antibiotic choice for spontaneous bacterial peritonitis. Clin Infect Dis 1999; 28: Zimhony O, Chmelnitsky I, Bardenstein R et al. Endocarditis caused by extended-spectrum beta-lactamase-producing Klebsiella pneumoniae: emergence of resistance to ciprofloxacin and piperacillin/tazobactam during treatment despite initial susceptibility. Antimicrob Agents Chemother 2006; 50:

Impact of MIC of piperacillin/tazobactam in the outcome of patients with. Running title: Piperacillin/tazobactam MIC in invasive ESBL-E.

Impact of MIC of piperacillin/tazobactam in the outcome of patients with. Running title: Piperacillin/tazobactam MIC in invasive ESBL-E. AAC Accepts, published online ahead of print on 22 April 2013 Antimicrob. Agents Chemother. doi:10.1128/aac.00135-13 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 3 4 5 6