ORIGINAL AND CLINICAL ARTICLES Anaesthesiology Intensive Therapy 2013, vol. 45, no 2, 67 72 ISSN 1642 5758 DOI: 10.5603/AIT.2013.0015 www.ait.viamedica.pl Comparison of in vitro efficacy of ertapenem, imipenem and meropenem by the Enterobacteriaceae strains family Aneta Guzek 1, Dariusz Tomaszewski 2, Zbigniew Rybicki 2, Andrzej Truszczyński 2, Mariusz Barański 3, Krzysztof Korzeniewski 4 1 Microbiology Research Unit, Department of Laboratory Diagnostics, Military Institute of Medicine in Warsaw, Poland 2 Department of Anaesthesiology and Intensive Therapy, Military Institute of Medicine in Warsaw, Poland 3 Ward of Musculoskeletal Infections, Department of Orthopaedics and Traumatology, Military Institute of Medicine in Warsaw, Poland 4 Department of Epidemiology and Tropical Medicine, Military Institute of Medicine in Warsaw, Poland ABSTRACT Background. The members of the bacterial Enterobacteriaceae family play an important role in the aetiology of many hospital infections. Some of them are able to produce β-lactamase, an enzyme which induces the resistance of those bacteria to penicillins, cephalosporins and, in severe infections, to penicillins with β-lactamase inhibitors. In this situation, the carbapenems become the drugs of choice. The objective of this study was to analyse the in vitro efficacy of three carbapenems: ertapenem, imipenem and meropenem against bacterial species of the Enterobacteriaceae family. Methods. A total of 99 bacterial species (including ten bacterial species producing the ESBL mechanism), isolated between September 2011 and March 2012 from diagnostic material collected from patients of surgical clinics In the department of motoskeletal system infections and the critical care unit, hospitalised in the Military Institute of Medicine in Warsaw, were analysed. The values of MIC 50 and MIC 90 were recorded. Results. All isolated bacterial species were susceptible to meropenem. One strain of Morganella morganii was resistant to imipenem, while one strain of Enterobacter cloaceae and one strain of Klebsiella pneumoniae were resistant to ertapenem. In the Enterobacteriaceae ESBL( ) group, the values of MIC 50 were 0.006 μg ml 1 for ertapenem, 0.19 μg ml 1 for imipenem, and 0.032 μg ml 1 for meropenem, and the MIC 90 values were: 0.25 μg ml 1, 0.5 μg ml 1 and 0.125 μg ml 1, respectively. In the Enterobacteriaceae ESBL(+) group, the values of MIC 50 were 0.38 μg ml 1, 0.25 μg ml 1, 0.064 μg ml 1, and the values of MIC 90 were 0.5 μg ml 1, 0.25 μg ml 1 and 0.125 μg ml 1, respectively. Conclusions. All analysed carbapenems had high in vitro efficacy against both Enterobacteriaceae ESBL( ) and Enterobacteriaceae ESBL(+) bacterial species. Key words: infections, bacteria, Enterobacteriaceae, antibiotics, ertapenem, imipenem, meropenem Anaesthesiology Intensive Therapy 2013, vol. 45, no 2, 67 72 Fermenting enteric bacilli of the Enterobacteriaceae family are responsible for a substantial proportion of hospital- -acquired urinary, abdominal, respiratory and wound infections, including chronic crural ulcerations. Enterobacteriaceae, Klebsiella pneumoniae and Escherichia coli, in particular, are capable of producing extended-spectrum beta-lactamases (ESBLs), enzymes that hydrolyze the β-lactam bond in the molecules of β-lactam antibiotics, which results in bacterial resistance to the penicillins, cephalosporins and penicillins with β-lactamase inhibitors in severe infections. Under such circumstances, the drugs of choice for the treatment of infections are the carbapenems [1]. The bacterial susceptibility to antibiotics, including carbapenems, is increasingly low. The susceptibility of Gram-negative bacteria to antibiotics varies considerably by geographical location. An analysis published in 2012 of 23918 Gram-negative bacteria isolated from ICU patients in six world regions in the years 2004 2009 demonstrated that the lowest percentage 67
Anaesthesiol Intensive Ther 2013, vol. 45, no 2, 67 72 of ESBL-producing Klebsiella pneumoniae and Escherichia coli strains was found in North America, 12.8% and 4.7%, respectively [2]. A high percentage of ESBL(+) Klebsiella pneumoniae was identified in Latin America (45.5%) and Africa (54.9%), and of ESBL(+) Escherichia coli in the Middle East (32.4%). In all regions, a high susceptibility (over 90%) of Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, Enterobacter cloacae and Serratia marcescens to imipenem was observed. All strains of Klebsiella oxytoca and Serratia marcescens were susceptible to meropenem. The susceptibility of Acinetobacter baumannii to meropenem was found to differ substantially depending on the region, ranging from 60.4% in North America to 15.9% in Africa; the susceptibility of Pseudomonas aeruginosa to meropenem varied from 79.1% in North America to 51.4% in Africa [2]. The antibiotic susceptibility of bacteria varies greatly between ICUs and surgical wards versus average hospital susceptibility, in favor of the former. The number of Enterobacteriaceae producing Klebsiella pneumoniae carbapenemase (KPC), Verona integron-encoded metallo-β-lactamase (VIM), IMP (imipenemase) and New Delhi metallo-β-lactamase (NDM-1) is increasing, additionally limiting the antibacterial treatment options. The above-mentioned strains play a significant role in hospital-acquired infections and in infections in long-term care facility patients [3]. The data from 35 European and Turkish ICUs published in 2009 revealed the following resistance of ESBL(+) Enterobacteriaceae to imipenem: 1.1% of Escherichia coli strains in Hungary, 1.1% of Klebsiella pneumoniae in Hungary and 13.6% in Turkey, 3.1% of Enterobacter cloacae in Sweden and 6.3% in Turkey, 10.6% of Pseudomonas aeruginosa in Romania and 48% in Turkey and 0% of Acinetobacter baumannii in Estonia and 91% in Malta [4]. The development of bacterial resistance is a significant problem in Poland. The differences in the in vitro susceptibility of bacteria to antibiotics, including the carbapenems, can be observed in individual hospitals and departments. In a 2010 study, the isolation of metallo-b-lactamase producing Klebsiella pneumoniae resistant to carbapenems was described [5]. Higher incidences of Acinetobacter baumannii isolates resistant to imipenem and meropenem were shown (2.7% in 2008 vs. 31% in 2009 and 2.1% in 2007 vs. 34.6% in 2009, respectively) [6]. Awareness of the microbiological setting of our departments and of the minimal inhibitory concentration (MIC) of the antibiotics effective against the pathogens isolated there can be essential for therapeutic decisions and planning for empirical antibiotic therapy. In many cases, the only therapeutic option in highly resistant Gram-negative bacteria-induced infections is the use of carbapenems. This group of antibiotics is characterized by low general toxicity. The aim of this study was to determine the in vitro susceptibility of Enterobacteriaceae to three carbapenem antibiotics, i.e., ertapenem, imipenem and meropenem. METHODS The specimens were collected between September 2011 and March 2012 from patients at the Military Institute of Medicine in Warsaw in the following departments: the General, Metabolic, Oncologic and Thoracic Surgery Department; the General, Oncologic, and Vascular Surgery Department; the Musculoskeletal Infections Ward; and the Intensive Care Unit. The specimens for bacterial identification included peritoneal and gallbladder fluid, abdominal swabs and swabs from wounds and diabetic foot ulcerations. The genus and species of the bacterial strains were identified using an automated analyzer (VITEK 2, BioMérieux, Marcy l Etoile, France), according to the manufacturer s instructions. The susceptibility of the isolates was determined based on the MIC. Testing strips with a predetermined concentration gradient of an antibiotic (Etest ; BioMérieux, Marcy l Etoile, France) were used to measure the range of concentration for ertapenem, imipenem and meropenem, which was 0.002 32 μg ml -1 on Mueller-Hinton medium (BioMérieux, Marcy l Etoile, France) (Fig. 1). The inoculated plates were incubated at 35 ± 2 C for 16 18 h in an oxygen atmosphere. The diagnostics were performed according to the recommendations of the National Reference Center for Microbial Drug-Susceptibility (NRCMDS). Escherichia coli ATCC 25922 was used for the quality control. The isolated ESBL-producing bacterial strains were tested to detect the mechanism of resistance. According to the NRCMDS recommendations, the double disc synergy test (DDST) was applied using Oxoid discs (Thermo Scientific, Great Britain) with reference antibiotics ceftazidime (30 μg), cefotaxime (30 μg) and a disc containing amoxicillin (20 μg) with a β-lactamase inhibitor clavulanic acid (10 μg) (Fig. 2). MIC 50 is the minimal antibiotic concentration required to inhibit the growth of 50 % of isolated bacterial strains (median). In an n-sized sample, MIC 50 equals n 0.5, when n is even; otherwise, MIC 50 corresponds to (n + 1) 0.5. MIC 90 is the minimal antibiotic concentration required to inhibit the growth of 90% of isolated bacterial strains [7]. The results obtained were presented for the entire study population, the surgical departments, the ICU and the Musculoskeletal Infections ward because of the specificity of the departments and ward. The data were archived and calculated using Microsoft Office 2010 (WIM license). MIC 50 and MIC 90 were calculated by determining the 50 th percentile (median) and 90 th percentile of the results. 68
Aneta Guzek et al., Carbapenems in Enterobacteriaceae infections Figure 1. Determination of the MIC using the disc with the antibiotic concentration gradient Figure 2. Double-disc synergy test (DDST) RESULTS The antibiotic susceptibility of 99 Enterobacteriaceae strains was analyzed, including 51 strains of Escherichia coli (51.52%), 14 of Enterobacter cloacae (14.14%), 12 of Klebsiella pneumoniae (12.12%), 7 of Morganella morganii (7.07%), 4 of Citrobacter freundii (4.04%), 5 of Klebsiella oxytoca (5.05%) and two of (2.02%) of Citrobacter braakii, Proteus spp and Serratia marcescens, each. In the isolated bacteria, the ESBL mechanism was identified in 58.33% (7 strains) of Klebsiella pneumoniae, 25% (1 strain) of Citrobacter freundii, 7.14% (1 strain) of Enterobacter cloacae, and 1.06% (1 strain) of Escherichia coli. The remaining analyzed bacteria (Citrobacter braakii, Klebsiella oxytoca, Morganella morganii, Proteus spp. and Serratia) did not induce the ESBL mechanism. The findings were interpreted based on the guidelines of the European Committee on Antibacterial Susceptibility Testing (EUCAST). The borderline values of the MIC according to which strains were classified as resistant/susceptible are presented in Table 1. The MIC 50 and MIC 90 of the carbapenems for ESBL( ) Enterobacteriaceae MIC 50 and for ESBL(+) Enterobacteriaceae are given in Tables 2 and 3, respectively, for all of the specimens and divided into the strains isolated from the specimens of the surgical, ICU and musculoskeletal infection patients. The efficacy of the carbapenems against the isolated Enterobacteriaceae was found to be extremely high. In the specimens, only one strain, i.e., Morganella morganii, was resistant to imipenem (MIC 3.0 μg ml -1 ), and one strain of Klebsiella pneumoniae and one strain of Enterobacter cloacae (MIC 1.0 μg ml -1 ) were resistant to ertapenem. The MIC 50 and MIC 90 for ESBL( ) Enterobacteriaceae and ESBL(+) Enterobacteriaceae are presented in Figs. 3 and 4, respectively. The comparison of the MIC 50 and MIC 90 for individual antibiotics at concentrations adopted by EUCAST as the upper limit of susceptibility (0.5 μg ml -1 for ertapenem and 2 μg ml 1 for imipenem and meropenem) demonstrated that the MIC 50 for ertapenem was 1.2% while the MIC 90 constituted 50% of the upper susceptibility limit. The respective values for imipenem were 9.5% and 25% and 1.6% and 6.25% for meropenem (Table 3). A similar tendency was observed in Table 1. Carbapenem MIC values for the Enterobacteriaceae family according to EUCAST guidelines Antibiotic Ertapenem (μg ml 1 ) Imipenem (μg ml 1 ) Meropenem (μg ml 1 ) Susceptibility 0.5 2.0 2.0 Resistance > 1.0 > 8.0 > 8.0 Table 2. Carbapenem MIC 50 and MIC 90 values (μg ml 1 ) for ESBL( ) Enterobacteriaceae Departments Antibiotic MIC 50 MIC 90 All ertapenem 0.006 0.25 imipenem 0.19 0.5 meropenem 0.032 0.125 ICU and WMSI ertapenem 0.008 0.38 imipenem 0.19 0.38 meropenem 0.047 0.094 Surgical wards ertapenem 0.006 0.25 imipenem 0.19 0.5 meropenem 0.032 0.125 ICU Intensive Care Unit; WMSI Musculoskeletal Infections Ward 69
Anaesthesiol Intensive Ther 2013, vol. 45, no 2, 67 72 the analysis of the specimens from the individual wards and departments. All the ESBL(+) Enterobacteriaceae strains were susceptible to the carbapenems (Table 4). The MIC values were compared with concentrations considered the upper limit of susceptibility (0.5 μg ml -1 for ertapenem and 2 μg ml -1 for imipenem and meropenem. The respective values were as follows: 76% and 100% for ertapenem, 12.5% and 12.5% for imipenem and 3.2% and 6.25% for meropenem (Table 5). A similar trend was observed in the specimens from the individual wards and departments. The findings are summarized in Table 6. DISCUSSION Enterobacteriaceae bacteria play an essential role in hospital-acquired infections, particularly in patients after abdominal surgical procedures; they are responsible for pneumonia, blood infections and urinary infections. In intensive care units in Poland, the major infection site is the abdominal cavity. Enterobacteriaceae were the etiological factor in 15.7% of the patients with blood infections [8]. Enterobacteriaceae are capable of developing numerous mechanisms of resistance such as ESBLs, KPCs or AmpC β-lactamase. Because of these mechanisms and the uncomplicated transmission of resistance, the bacteria in question constitute a substantial epidemiological hazard. The in vitro tests were performed using modern diagnostic methods in a certified laboratory, which guarantees the reliability of the results. Among 99 isolated pathogens, 9.9% were bacteria with the ESBL resistance mechanism. This percentage is markedly lower than the percentage of resistance in the United States where, in 2006, this mechanism was observed in 31% of the Enterobacter cloacae strains and 13% of the Pseudomonas aeruginosa strains isolated from intensive care unit patients [9]. Table 3. Carbapenem MIC 50 and MIC 90 (μg ml 1 ) values for ESBL(+) Enterobacteriaceae Wards Antibiotic MIC 50 MIC 90 All ertapenem 0.38 0.5 imipenem 0.25 0.25 meropenem 0.064 0.125 ICU and WMSI ertapenem 0.38 0.5 imipenem 0.19 0.25 meropenem 0.064 0.125 Surgical wards ertapenem 0.25 0.5 imipenem 0.25 0.25 meropenem 0.047 0.75 ICU Intensive Care Unit; WMSI Musculoskeletal Infections Ward Figure 3. MIC 50 and MIC 90 for ESBL( ) Enterobacteriaceae Figure 4. MIC 50 and MIC 90 for ESBL(+) Enterobacteriaceae Table 4. Carbapenem MIC 50 and MIC 90 values for ESBL( ) Enterobacteriaceae in relationship to the highest antibiotic concentration, considered the drug-susceptibility cut-off point according to EUCAST. Collective data for all the bacterial strains without division into individual wards and units Antibiotic MIC 50 MIC 90 ertapenem 1.2% 50% imipenem 9.5% 25% meropenem 1.6% 6.25% Table 5. Carbapenem MIC 50 and MIC 90 values for ESBL(+) Enterobacteriaceae in relationship to the highest antibiotic concentration considered the drug-susceptibility cut-off point according to EUCAST. Collective data for all bacterial strains without division into individual wards and units Antibiotic MIC 50 MIC 90 ertapenem 76% 100% imipenem 12.5% 12.5% meropenem 3.2% 6.25% 70
Aneta Guzek et al., Carbapenems in Enterobacteriaceae infections Carbapenems comprise the only group of antibiotics with low general toxicity for treating infections with ESBL (+) Enterobacteriaceae, and they should be used under strict controls. In this paper, an attempt was made to determine whether individual carbapenems (ertapenem, imipenem and meropenem) showed similar in vitro efficacy in inhibiting Enterobacteriaceae growth. The susceptibility of the bacteria was assessed using E-tests, which are considered to be the most reliable, assuming that borderline concentrations were susceptible (following the EUCAST guidelines). Table 2 demonstrates that, except for three strains, all of the bacteria were susceptible to low concentrations of carbapenems. At the antibiotic concentration reaching 25% of the MIC, ertapenem, imipenem and meropenem inhibited 75.8%, 92% and 99%, respectively, of the susceptible bacteria. The highest efficacy was observed for meropenem, followed by imipenem and ertapenem (markedly lower). The comparison of our data with the results of the European study on bacterial susceptibility [10] shows that bacterial resistance in our center is low. To analyze the problem more objectively, the MIC 50 and MIC 90 values were compared with the MIC values of antibiotics considered borderline for a given strain to be susceptible to a particular drug (EUCAST). It appears that MIC 90 is more useful for assessing the efficacy of bacterial growth inhibition. The data presented in Tables 5 and 6 reveal that in ESBL( ) bacteria at MIC 90, imipenem and meropenem inhibit the growth of bacteria at lower concentrations than ertapenem. This observation is to some extent consistent with the results of the MIC distribution analysis. It is difficult to assess explicitly this phenomenon because there are no data in the literature comparing the MIC values with MIC 50 and MIC 90, Table 6. Distribution of the MICs of the individual antibiotics MIC value (μg ml 1 ) 0.023 0.004 0.006 0.008 0.012 0.016 0.019 0.023 0.025 0.032 0.038 0.047 0.06 0.064 0.094 0.12 0.125 0.19 0.25 0.38 0.5 0.75 1.0 1.5 3.0 All wards incidence 16 28 15 7 5 1 5 1 2 1 1 1 3 4 3 4 2 percentage 16.16 28.28 15.15 7.07 5.05 1.01 5.05 1.01 2.02 1.01 1.01 1.01 3.03 4.04 3.03 4.04 2.02 incidence 2 4 3 3 1 1 2 1 1 1 1 percentage 10.00 20.00 15.00 15.00 5.00 5.00 10.00 5.00 5.00 5.00 5.00 ICU and WMSI Surgical incidence 14 24 12 4 4 3 2 1 1 3 4 2 3 2 percentage 17.72 30.38 15.19 5.06 5.06 3.80 2.53 1.27 1.27 3.80 5.06 2.53 3.80 2.53 All wards incidence 1 21 41 18 7 4 6 1 percentage 1.01 21.21 41.41 18.18 7.07 4.04 6.06 1.01 incidence 3 7 4 4 1 1 percentage 15.00 35.00 20.00 20.00 5.00 5.00 ICU and WMSI Surgical incidence 1 18 34 14 3 3 5 1 percentage 1.27 22.78 43.04 17.72 3.80 3.80 6.33 1.27 All wards incidence 1 3 10 26 21 12 9 3 3 6 4 1 percentage 1.01 3.03 10.10 26.26 21.21 12.12 9.09 3.03 3.03 6.06 4.04 1.01 incidence 1 4 2 3 5 2 1 1 1 percentage 5.00 20.00 10.00 15.00 25.00 10.00 5.00 5.00 5.00 ICU and WMSI Ertapenem Imipenem Meropenem Surgical incidence 1 2 6 24 18 7 7 2 2 5 4 1 percentage 1.01 2.53 7.59 30.38 22.78 8.86 8.86 2.53 2.53 6.33 5.06 1.27 ICU Intensive Care Unit; WMSI Musculoskeletal Infections Ward; MIC values above the cut-off point for susceptible strains were marked in gray (according to EUCAST) 71
Anaesthesiol Intensive Ther 2013, vol. 45, no 2, 67 72 and the findings cannot be applied to clinical practice. This issue requires further study. The efficacy of antibiotics depends not only on the susceptibility of bacteria but also on the time of administration of an appropriate dose and the penetration of the drug to the target infection site [11]. In a number of cases, e.g., severe sepsis or septic shock, despite following the principles of antibiotic therapy, the antibiotic concentration in the tissues is lower than the MIC. This low concentration is caused by impaired distribution of the drug for the following reasons: transfusions of large volumes of fluids; the use of catecholamines, which constrict the vessels and hinder drug penetration; and enhanced excretion by the kidneys, through which the blood flow in the hyperdynamic phase of shock is increased [12, 13]. In the clinical states described, it is essential for the MIC to be as low as possible because the drug concentration required for antibiotic susceptibility can be the element that compensates for impaired distribution of antibiotics to the tissues. These limited findings of our study should be emphasized, although additional work is required to draw diagnostically useful and more clinically significant conclusions. CONCLUSIONS 1. Ertapenem, imipenem and meropenem show extremely high in vitro efficacy for inhibiting the growth of ESBL( ) Enterobacteriaceae and ESBL(+) Enterobacteriaceae ESBL at low MIC values. 2. The comparison of the MIC 50 and MIC 90 levels of the antibiotics analyzed demonstrates that meropenem is most effective, followed by imipenem and ertapenem; diagnostically and clinically significant conclusions should not be drawn based on the above observations. MIC values above the cut-off point for the susceptible strains were marked in gray (according to EUCAST). References: 1. Zhanel GG, Wiebe R, Dilay L, et al.: Comparative review of the carbapenems. Drugs 2007; 67: 1027 1052. 2. Bertrand X, Dowzicky MJ: Antimicrobial susceptibility among gram- -negative isolates collected from intensive care units in North America, Europe, the Asia-Pacific rim, Latin America, the Middle East, and Africa between 2004 and 2009 as part of the tigecycline evaluation and surveillance trial. Clin Ther 2012; 34: 124 137. 3. Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL: Carbapenemases in klebsiella pneumoniae and other enterobacteriaceae: an evolving crisis of global dimensions. Clin Microbiol Rev 2012; 25: 682 707. 4. Hanberger H, Arman D, Gill H, et al.: Surveillance of microbial resistance in european intensive care units: A first report from the Care-ICU programme for improved infection control. Intensive Care Med 2009; 35: 91 100. 5. Sekowska A, Gospodarek E, Kruszyńska E, et al.: First isolation of metalloβ-lactamase producing Klebsiella pneumoniae strain in Poland. Anaesthesiol Intensive Ther 2010; 42: 25 27. 6. Bogiel T, Kwiecińska-Piróg J, Jachna-Sawicka K, Gospodarek E: Szczepy Acinetobacter baumannii oporne na karbapenemy. Med Dosw Mikrobiol 2010; 62: 119 126. 7. Schwarz S, Silley P, Simjee S, et al.: Editorial: Assessing the antimicrobial susceptibility of bacteria obtained from animals. J Antimicrob Chemother 2010; 65: 601 604. 8. Kübler A, Durek G, Zamirowska A, et al.: Severe sepsis in Poland results of internet surveillance of 1043 cases. Med Sci Monit 2004; 10: CR635 641. 9. Gupta N, Limbago BM, Patel JB, Kallen AJ: Carbapenem-resistant enterobacteriaceae: Epidemiology and prevention. Clin Infect Dis 2011; 53: 60 67. 10. Drusano GL: Antimicrobial pharmacodynamics: Critical interactions of 'bug and drug. Nat Rev Microbiol 2004; 2: 289 300. 11. Bochud PY, Bonten M, Marchetti O, Calandra T: Antimicrobial therapy for patients with severe sepsis and septic shock: an evidence-based review. Crit Care Med 2004; 32: S495 512. 12. Joukhadar C, Frossard M, Mayer BX, et al.: Impaired target site penetration of beta-lactams may account for therapeutic failure in patients with septic shock. Crit Care Med 2001; 29: 385 391. 13. Brunner M, Pernerstorfer T, Mayer BX, Eichler HG, Müller M: Surgery and intensive care procedures affect the target site distribution of piperacillin. Crit Care Med 2000; 28: 1754 1759. Corresponding author: Dariusz Tomaszewski, MD, PhD Department of Anaesthesiology and Intensive Therapy, Military Institute of Medicine in Warsaw ul. Szaserów 128, 04 141 Warszawa, Poland tel.: + 48 22 681 68 95 e-mail: dtomaszewski@wim.mil.pl Received: 30.10.2012 Accepted: 23.01.2013 72