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Antimicrobial agents active against carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates in Lebanon

Antimicrobial agents active against carbapenem-resistant Escherichia coli and Klebsiella... Introduction: It is not yet clear which antimicrobial agents should be used to treat the ominously increasing infections with carbapenem-resistant (CR) bacteria. We therefore investigated the activity of different antimicrobial agents against CR Escherichia coli and Klebsiella pneumoniae in Lebanon. Methodology: This retrospective study assessed the minimum inhibitory concentrations (MICs) of three carbapenems (by Etest), as well as the in vitro activity of eight other antimicrobials (by disk diffusion) against CR E. coli (n = 300) and K. pneumoniae (n = 232) isolates recovered at a major University Medical Center in Lebanon. Results: Higher percentages of isolates showing carbapenem MICs of ≤ 8 µg/mL were noted among the CR E. coli compared to the CR K. pneumoniae for ertapenem (48% vs 27%), imipenem (74 % vs 58%) and meropenem (82% vs 63%). Among the eight other antimicrobials, activity was generally higher when the MICs for the three carbapenems were ≤ 8 µg/mL. Regardless of the MIC level of the three carbapenems, very low susceptibility rates (≤ 33%) were noted for ciprofloxacin, trimethoprim-sulfamethoxazole and aztreonam against both E. coli and K. pneumoniae isolates. With Amikacin, higher susceptibility rates were seen against E. coli isolates (81%-97%) than against K. pneumoniae isolates (55%-86%), also reflecting higher activity than gentamicin (44%-54%). The best activity (66%-100%) was observed for tigecycline, colistin and fosfomycin against both CR species. Conclusions: Based on the in vitro findings in this study, the combination of a carbapenem showing an MIC of ≤ 8 µg/mL together with an active colistin, tigecycline, or fosfomycin, would offer a promising treatment option for patients infected with CR E. coli or K. pneumoniae. Key words: Carbapenem-resistance; E. coli; K. pneumoniae; in vitro testing; antimicrobial resistance; Lebanon. J Infect Dev Ctries 2018; 12(3):164-170. doi:10.3855/jidc.9279 (Received 31 August 2017 – Accepted 16 January 2018) Copyright © 2018 Araj et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction increasing health costs. This situation is a major Globally, infection with carbapenem resistant (CR) challenge for the treating clinicians and infection Escherichia coli (Ec) and Klebsiella pneumoniae (Kp) control professionals [1-3,7,8]. In Lebanon, similar as well as other CR Enterobacteriaceae (CRE) had been problems and challenges have been encountered since on the rise, often accompanied by high rates of CRE pathogens were introduced in this country several resistance to a wide range of antimicrobials. These years ago [9,10]. As a result, several studies have been pathogens, together with the dwindling antimicrobial conducted to describe the phenotypic and molecular armament, constitute a most concerning contemporary features of CR E. coli and K. pneumoniae in this threat to health in many countries [1-3]. The Center for country [5,11-15]. Disease Control and Prevention in the USA noted an Because of the very limited available options to increase in the percentages of CRE from 1.2% in 2001 treat CR isolates, this study was undertaken to identify to 4.2% in 2011, where Klebsiella spp. accounted for the types and percentages of antimicrobial agents active the highest increase in proportion, a rise from 1.6% to against CR E. coli and K. pneumoniae resistant to 10.4% [4]. In Lebanon, a notable increase was observed ertapenem, imipenem, and/or meropenem. at our medical center in the percentages of CR, from 2010 to 2016 for Ec (0.1% to 5%) and Kp (0.7% to 8%), Methodology as well as in other hospitals [5,6]. Bacterial Isolates The ominous spread of these pathogens together Consecutive non-duplicate isolates of carbapenem with the scarcity of treatment options results in a serious resistant E. coli (300 isolates) and K. pneumoniae (232 health impact, seen in high rates of morbidity and isolates) at the Clinical Microbiology Laboratory, mortality (up to 75% of infected patients) and Department of Pathology and Laboratory Medicine, Araj et al. – Active agents against CRE J Infect Dev Ctries 2018; 12(3):164-170. American University of Beirut Medical Center Bank # 0346, specified colistin MIC of 4 µg/mL)] were (AUBMC) during the period March 2008 to June 2016 also used to ensure the quality of testing. were investigated. Identification of the isolates was based on standard biochemical methods [5]. Statistical Analysis The source distribution of these E. coli and K. Our data was analyzed using Stata v. 13 software pneumoniae isolates was: urine (39% and 32%), body Package (Stata Corp LP, College Station, Texas, USA) screen (19% and 23%), wound/pus/abscess (17% and for determining the p value, considering less than ≤ 0.05 18%), blood (10% and 9%), respiratory (9% and 5%), as significant. and other fluids (6% and 13%). Results Antimicrobial Susceptibility Testing The MICs (µg/mL) for ertapenem, imipenem and The minimal inhibitory concentrations (MICs) of meropenem against the CR E. coli (n = 300) and K. ertapenem, imipenem and meropenem were determined pneumoniae (n = 232) isolates were 8 and 24, 2 and 4 using the Etest methodology (AB BIODISK, Solna, and 1 and 4, respectively. The MICs (µg/mL) for these Sweden) according to the manufacturer’s guidelines. carbapenems were ≥ 32 ug/mL against both E. coli and The span of MIC levels on these strips ranges between K. pneumoniae isolates. ≤ 0.025 and ≥ 32 µg/mL. The 2016 Clinical and The distribution of the MICs (µg/mL) for Laboratory Standards Institute (CLSI) MICs’ ertapenem, imipenem and meropenem among these CR breakpoints (µg/mL) for Enterobacteriaceae were used E. coli and K. pneumoniae isolates are shown in Figures to interpret the susceptibility category as susceptible, 1 and 2. The MIC (µg/mL) level for the three intermediate and resistant, respectively; for ertapenem: carbapenems ranged between ≤ 0.25 and ≥ 32. The 0.5, 1, 2; for imipenem: 1, 2, 4; for meropenem: 1, 2, 4 [16]. Figure 1. Distribution of carbapenem MICs among E. coli isolates (n = 300). The disk diffusion (DD) antimicrobial susceptibility testing (except for colistin) was done and interpreted according to the CLSI standards [16]. Commercial disks (BBL, Becton Dickinson, USA) were used, with antimicrobial concentrations of: amikacin 30 g, gentamicin 10 g, ciprofloxacin 5 g, trimethoprim/sulfamethoxazole 1.25/23.75 g, tigecyline 15 g, colistin 10 g, fosfomycin/trometamal (200 μg), and aztreonam 30 g. Colistin disk diffusion was used and interpreted according to the study of Gelani et al. [17], where the susceptible, intermediate and resistant zone of inhibition were ≤ 11 mm, 12-13 mm, and ≥ 14 mm, respectively. For screening of carbapenemase-producers, Figure 2. Distribution of carbapenem MICs among K. ertapenem disks (10 g) were used. Those isolates pneumoniae isolates (n = 232). showing < 25 mm zone of inhibition were then tested by Etest to confirm the MICs of ertapenem, imipenem and meropenem. Quality Control The American Type Culture Collection (ATCC) quality control strains of E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were used to ensure correct performance of the Etest and disk diffusion methods. When received in February 2016, the quality control reference strains for colistin [CDC E. coli reference strains MCR-1 (AR-Bank # 0349, specified colistin MIC of 2-4 µg/mL) and MCR-1 (AR- 165 Araj et al. – Active agents against CRE J Infect Dev Ctries 2018; 12(3):164-170. figures reflect the distribution of the MICs up to ≥ 12 K. pneumoniae, regardless of the MICs levels of µg/mL and not up to ≥ 32 µg/mL due to space ertapenem, imipenem and meropenem (Table 1). limitation. The distribution of the CR E. coli and K. Discussion pneumoniae isolates according to their overall The clinical microbiology laboratory (CML) has a carbapenems MICs, as well as carbapenems MICs of ≤ key role in promptly detecting and reporting multi-drug 8 or ≥ 8 µg/mL in relation to their susceptible rates to resistant (MDR) pathogens to guide targeted other antimicrobial agents, namely: amikacin, antimicrobial therapy, which will influence outcome. In gentamicin, ciprofloxacin, trimethoprim- this context, the CML at AUBMC reported the first alert sulfamethoxazole, tigecycline, colistin, fosfomycin and about CRE in Lebanon, when the first imipenem aztreonam are presented in Table1. resistant K. pneumoniae that harbors the bla-OXA-48 Three of the antimicrobial agents tested, gene was recovered from the urine of an 8-year- old girl ciprofloxacin, trimethoprim-sulfamethoxazole and in 2008 [9]. aztreonam, showed very low susceptibility rates (≤ Two years later (July 2010), three Iraqi patients 33%) regardless of the MICs level against both E. coli presented at our medical center seeking treatment. CR and K. pneumoniae isolates, and with little difference E. coli and K. pneumoniae were isolated from different among the three carbapenems. Among specimens of these patients, and the isolates harbored aminoglycosides, the susceptibility rates of gentamicin not only the bla-OXA-48 gene but also the very were low, ranging between 44% and 54%, for both E. threatening bla-NDM-1 gene, producing the novel New coli and K. pneumoniae isolates, and with little Delhi Metallo ß- lactamase (MBL) gene [10]. difference among the 3 carbapenems. On the other Subsequently, the incidence of CR Enterobacteriaceae hand, the E. coli isolates showed higher susceptibility continued to rise [5]. Such critically resistant pathogens rates to amikacin (range: 81% to 97%), compared to the warranted publishing an overview to inform the K. pneumoniae isolates (range: 55% and 86%), as noted medical and paramedical community on the local and for the three carbapenems and regardless of the MICs regional epidemiology of carbapenem resistance, the category. Tigecycline, colistin and fosfomycin showed mechanisms involved, screening and detection high activity (66%-100%) against both CR E. coli and methods, as well as their treatment and control [11]. Table 1. Distribution of CR E. coli (n = 300) and K. pneumoniae (232) isolates according to carbapenem MICs in relation to susceptibility rates (%) of eight other antimicrobial agents. Carbapenems & Percent (%) of susceptible isolates to antimicrobial agents CRE Ħ Ħ Ħ MICs (µg/mL) AMK GENT CIP SXT TIGE COLI FOSFO AZT Ertapenem ≤ 8 E. coli 97* 53 28 16 100 82 89 20* > 8 E. coli 88 47 11 16 99 86 93 10 All E. coli 89 51 19 16 99 84 89 15 ≤ 8 K. pneumoniae 84* 40 33 27 87 94* 86* 30* > 8 K. pneumoniae 68 49 24 21 92 79 67 23 All K. pneumoniae 73 47 15 23 90 82 73 25 Imipenem ≤ 8 E. coli 94* 48 19 14 100 89* 94* 15 > 8 E. coli 85 54 16 23 98 74 87 16 All E. coli 92 53 19 17 99 84 89 15 ≤ 8 K. pneumoniae 86* 44 29 24 89 94* 83* 25 > 8 K. pneumoniae 57 49 21 21 94 73 61 24 All K. pneumoniae 77 48 26 23 93 83 68 25 Meropenem ≤ 8 E. coli 94* 49 21* 17 100 85 92* 15 > 8 E. coli 81 52 8 12 98 82 86 13 All E. coli 92 51 19 15 99 83 91 14 ≤ 8 K. pneumoniae 86* 45 31 25 89 88* 76* 30 > 8 K. pneumoniae 55 51 16 19 94 76 61 23 All K. pneumoniae 73 47 26 24 92 84 66 25 CR: Carbapenem resistant; AMK: amikacin; GEN: gentamicin; CIP: ciprofloxacin; SXT: trimethoprim-sulfamethoxazole; TIGe: tigecycline; COLI: colistin; FOSFO: fosfomycin; AZT: aztreonam; *p value significance ≤ 0.05 determined between MICs ≤ 8µg/mL and > 8µg/mL; The number of E. coli and K. pneumoniae isolates tested, respectively, were: tigecycline (198 and 160 isolates), colistin (156 and 125 isolates), fosfomycin (124 and 68 isolates). 166 Araj et al. – Active agents against CRE J Infect Dev Ctries 2018; 12(3):164-170. Generally, physicians rely on the in vitro carbapenemase (KPC) and OXA-48 -producers in K. antimicrobial susceptibility results of pathogens to pneumoniae isolates remain susceptible to gentamicin, provide optimum therapy to their patients. Empirical while this is rare for New Delhi metallo beta-lactamase combination therapy for CRE infected patients is (NDM) - producers. An in vitro synergistic effect was usually based on the local resistance epidemiology. observed when netilmicin and fosfomycin were tested Definitive therapy, however, should be guided by against a wide range of resistant pathogens. Also, a determination of the in vitro antimicrobial susceptibility combination of carbapenems and aminoglycosides profiles especially when considering combined showed in vitro and in vivo (in animals) synergistic treatment for CRE pathogens. This is very important effects against some KPC-producing K. pneumoniae since only very few drugs remain as last-resort agents isolates, but studies with carbapenemases other than (e.g. colistin, fosfomycin, tigecycline, and KPC are lacking [18,27]. A lack of effect was noted aminoglycosides) [18-22]. when a combination of gentamicin and fosfomycin was In our study, the in vitro activity of these last-resort tested against KPC-2-producing K. pneumoniae. In our agents against the CR E. coli and K. pneumoniae current study, the overall higher activity of amikacin isolates detected at our institution are the core of the compared to that of gentamicin against both CR Ec and following discussion. These results will be considered Kp may be attributed to the type of carbapenemase gene especially in relation to updates reported in the recent involved. For example, in the clinical setting, a study scientific literature [18,19]. from Greece reported a favorable outcome in five Colistin has significant activity against various patients infected with KPC-2-producing K. pneumoniae carbapenemase-producing isolates and is often used in who were treated with aminoglycoside plus colistin combination therapy (e.g., with aminoglycosides, (plus tigecycline in 2 patients) [29]. aztreonam, carbapenems, rifampin, tigecycline, or Tigecycline has also been recommended as part of fosfomycin) with a high (82.1%) clinical cure rate the initial combination regimen in the treatment of [18,23,24]. Caution in its use is advised, to avoid patients with CRE infections in different body sites, development of colistin-resistant Ec & Kp. In our study, other than UTI, especially when tigecycline MIC is ≤ 1 the overall high susceptibility rates (82%-84 % ) of both mg/L (the typical MICs was 0.5 mg/L for E. coli and Ec and Kp to colistin makes it an important first option 1–2 mg/L for K. pneumoniae). The overall crude to use in the treatment of such CR infections (Table 1). mortality when treating with tigecycline was higher Fosfomycin has also been considered as part of a with monotherapy than with combined treatment (40– combination regimen (including at least one more 80% vs 0–33%) [18]. In our study, tigecycline showed active agent, e.g. with tigecycline and colistin) in the very high in vitro activity against both CR Ec and Kp, treatment of invasive infections with CRE pathogens making it an excellent agent in combined treatment [25]. Its use in combinations, however, resulted in options for such CR pathogens. Low in vitro resistance varied synergistic activity against CRE depending on rates against tigecycline were also reported in a 2008 the type of pathogen and the antibiotics used [26]. For study from our institution, in MDR and ESBL Ec (0%) example, Samonis et al. [27] reported that the and Kp (3%) [29]. The very low rates noted in the synergistic effect of fosfomycin was 55% to 79% with previous and current studies indicate a sustainable carbapenems, 7.1% to 36% with colistin, 21% to 30% activity of tigecycline against resistant isolates at our with tigecycline, and 25% to 43% with gentamicin. In center. this study, fosfomycin showed high activity (89-91%) Aztreonam testing was included in this study against the CR Ec, while the susceptibility of CR Kp because MBLs can hydrolyze carbapenems and all the was lower (66-73%). We conclude that fosfomycin can available beta-lactams with the exception of aztreonam. remain a viable option for the treatment of CR Whether aztreonam remains an option for treating pathogens especially Ec (Table 1). These levels are infections due to MBL producers that test susceptible to close to those we reported in 2012 on the fosfomycin this agent remains to be determined; currently there is susceptibility of ESBL-producing pathogens, where its no clinical experience with aztreonam for the treatment activity was higher against ESBL-producing E. coli of invasive infections due to CRE [30]. In our study, isolates (86%) than against K. pneumoniae isolates however, a very low aztreonam susceptibility (14-25%) (62%) [28]. was shown by both CR Ec and CR Kp isolates, Aminoglycosides have been used in combination indicating that the majority of these isolates were not with other classes of antibiotics with different results MBL producers. An earlier study on the CR genes [18,27]. For example, some K. pneumoniae involved in K. pneumoniae and E. coli isolates from our 167 Araj et al. – Active agents against CRE J Infect Dev Ctries 2018; 12(3):164-170. center indicated the prevalence of bla-OXA-1, bla- In our study the carbapenem rates of MIC ≤ 8 mg/L CTXM-15, bla-TEM-1, bla-CMY-2, bla-OXA-48, and for ertapenem, imipenem and meropenem were higher porin channel genes, and few had the NDM-1 gene [12]. when tested against E. coli: 48%, 74% and 82%, The antimicrobial agents discussed above have respectively, than against K. pneumoniae: 27%, 58% been considered for possible clinical relevance to treat and 63%, respectively. Such rates, especially for patients infected with CRE. The question of the imipenem and meropenem, in combination with the superior effectiveness of mono- or combined other in vitro active agents, can provide an adequate antimicrobial treatment of patients infected with CRE option in treating patients, particularly those with CR E. pathogens was excellently reviewed by Rodríguez- coli infection. Baño and colleagues in Spain and Karaiskos and Studies at our medical center since 2008 have been Giamarellou in Greece, looking at microbiological addressing molecular characterizations, resistance diagnosis and treatment of infections with mechanisms, genes involved, and other aspects of CR carbapenemase-producing Enterobacteriaceae [18,19]. Ec and Kp isolates [9,10,12-14]. The first CRE detected These comprehensive studies suggest a more favorable in Lebanon was a K. pneumoniae recovered from a 7- outcome (e.g. reduced mortality) using a combined year-old female child, which was characterized by PCR antimicrobial treatment for patients infected with CRE experiments using primers for multiple β-lactamase and pathogens. Besides, combined therapy may maximize carbapenemase genes: TEM, SHV, CTX-M, GES, bacterial killing (synergistic effect) and minimize KPC, IMI, OXA-1 ⁄ 2 ⁄ OXA-10 ⁄ 18, OXA-23 ⁄ 58, IMP, bacterial resistance. For example, Tumbarello et al. VIM, SPM, GIM and SIM. The only positive result was 2012 [31] reported a higher mortality rate among for a blaOXA-48-like gene [9]. Interestingly, this was patients treated with monotherapy compared to only the fourth report about this gene worldwide. combined therapy (54.3% vs 34.1%); the combination Subsequently, increasing numbers of CRE have been included tigecycline, colistin, and meropenem. noticed; some harbored the very threatening bla NDM- In addition, the inclusion of a carbapenem in the 1 gene, detected for the first time in Lebanon, in E. coli combined treatment showed promising results. For and K. pneumoniae isolates recovered from Iraqi example, Qureshi et al., reported lower mortality when patients coming for medical care at AUBMC [10]. carbapenem was used in combination with colistin or The underlying mechanisms and genes responsible tigecycline, compared to a combination that lacked a for the carbapenem-resistance in K. pneumoniae and E. carbapenem (12.5% vs 66.7%) [32]. Moreover, the coli revealed the presence of different Beta-lactamase effect of carbapenems in the combination was gene profiles including: bla-OXA-1, bla-CTXM-15, noticeable if the carbapenem MIC was ≤ 8 mg/L, and it bla-TEM-1, bla-CMY-2, bla-OXA-48 and NDM-1 was added to one or two in vitro fully active drugs genes in both genera; in addition, the K. pneumoniae (including colistin, tigecycline, an aminoglycoside or isolates were found to lack outer membrane porin fosfomycin) for such infections. Carbapenems, despite (OmpC and OmpF) encoding genes, while E. coli being hydrolyzed by carbapenemases, may retain some harbored these porin genes [12]. In addition, the activity against carbapenemase-producing isolates prevalence of carbapenem resistance encoding genes [18,19,33,34]. Such findings were vividly reflected in and their correlation with corresponding MIC against the study by Daikos et al. [20] who reported a lower ertapenem, imipenem and meropenem were studied mortality rate with treatment by a carbapenem- among CR E. coli (n=76) and CR K. pneumoniae containing combination with another active drug, such (n=54) isolates. The prevalence of blaOXA-48, as an aminoglycoside, colistin or tigecycline, than with blaNDM-1, blaTEM-1 and blaCTX-M-15 among the a combination without a carbapenem (19.3% vs 30.6%). E. coli isolates were 36%, 12%, 20% and 80%, It was important that the carbapenem had a MIC ≤ 8 respectively, while among K. pneumoniae isolates they mg/L [20]. Akova et al. found similar results [35]. were 37%, 28%, 28% and 72%, respectively. The Interestingly, the efficacy of using dual carbapenem presence of more than one carbapenem resistance (i.e., meropenem plus ertapenem) therapy has been encoding gene and/ or ESBL encoding gene did not shown in animal model of infection (i.e., mouse have an effect on the MIC value in K. pneumoniae pneumonia) and in humans infected with KPC isolates, while in E. coli they resulted in higher MIC producers. The suggested explanation was that values [14]. ertapenem most likely acts as a “suicide” molecule for We also assessed the effects of antimicrobial carbapenemase activity, whereas the more active drug, combination therapy against bacteria with different CR meropenem, retains its efficacy [36]. genes using BALB/c mice [13]. The mice were injected 168 Araj et al. – Active agents against CRE J Infect Dev Ctries 2018; 12(3):164-170. 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GS (2014) Antibiotic treatment of infections due to 36. Giamarellou H, Galani L, Baziaka F, Karaiskos I (2013) carbapenem-resistant Enterobacteriaceae: Systematic Effectiveness of double-carbapenem regimen for infections in evaluation of the available evidence. Antimicrob Agents humans due to carbapenemase-producing pandrug-resistant Chemother 58: 654–663. Klebsiella pneumoniae. Antimicrob Agents Chemother 57: 23. Temkin E, Adler A, Lerner A, Carmeli Y (2014) Carbapenem- 2388–2390. resistant Enterobacteriaceae: Biology, epidemiology, and management. Ann N Y Acad Sci 1323: 22–42. Corresponding author 24. Michalopoulos AS, Tsiodras S, Rellos K, Mentzelopoulos S, George F Araj, PhD, D(ABMM), FAAM Falagas ME (2005) Colistin treatment in patients with ICU- Professor and Director of Clinical Microbiology acquired infections caused by multiresistant Gram-negative Department of Pathology & Laboratory Medicine bacteria: The renaissance of an old antibiotic. Clin Microbiol American University of Beirut Medical Center Infect 11: 115–121. PO Box 11-0236, Beirut, Lebanon 1107-2020 25. Pontikis K, Karaiskos I, Bastani S, Dimopoulos G, Kalogirou Phone: +(961) -1- 350 000 ext 5215 M, Katsiari M, Oikonomou A, Poulakou G, Roilides E, Fax: +(961) -1- 370 845 Giamarellou H (2014) Outcomes of critically ill intensive care Email: garaj@aub.edu.lb unit patients treated with fosfomycin for infections due to pandrug-resistant and extensively drug resistant Conflict of interests: No conflict of interests is declared. carbapenemase-producing Gram-negative bacteria. Int J Antimicrob Agents 43: 52–59. 26. Falagas ME, Kastoris AC, Kapaskelis AM, Karagoergopoulos DE (2010) Fosfomycin for the treatment of multidrug-resistant, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infection in Developing Countries Unpaywall

Antimicrobial agents active against carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates in Lebanon

The Journal of Infection in Developing CountriesMar 31, 2018

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1972-2680
DOI
10.3855/jidc.9729
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Abstract

Introduction: It is not yet clear which antimicrobial agents should be used to treat the ominously increasing infections with carbapenem-resistant (CR) bacteria. We therefore investigated the activity of different antimicrobial agents against CR Escherichia coli and Klebsiella pneumoniae in Lebanon. Methodology: This retrospective study assessed the minimum inhibitory concentrations (MICs) of three carbapenems (by Etest), as well as the in vitro activity of eight other antimicrobials (by disk diffusion) against CR E. coli (n = 300) and K. pneumoniae (n = 232) isolates recovered at a major University Medical Center in Lebanon. Results: Higher percentages of isolates showing carbapenem MICs of ≤ 8 µg/mL were noted among the CR E. coli compared to the CR K. pneumoniae for ertapenem (48% vs 27%), imipenem (74 % vs 58%) and meropenem (82% vs 63%). Among the eight other antimicrobials, activity was generally higher when the MICs for the three carbapenems were ≤ 8 µg/mL. Regardless of the MIC level of the three carbapenems, very low susceptibility rates (≤ 33%) were noted for ciprofloxacin, trimethoprim-sulfamethoxazole and aztreonam against both E. coli and K. pneumoniae isolates. With Amikacin, higher susceptibility rates were seen against E. coli isolates (81%-97%) than against K. pneumoniae isolates (55%-86%), also reflecting higher activity than gentamicin (44%-54%). The best activity (66%-100%) was observed for tigecycline, colistin and fosfomycin against both CR species. Conclusions: Based on the in vitro findings in this study, the combination of a carbapenem showing an MIC of ≤ 8 µg/mL together with an active colistin, tigecycline, or fosfomycin, would offer a promising treatment option for patients infected with CR E. coli or K. pneumoniae. Key words: Carbapenem-resistance; E. coli; K. pneumoniae; in vitro testing; antimicrobial resistance; Lebanon. J Infect Dev Ctries 2018; 12(3):164-170. doi:10.3855/jidc.9279 (Received 31 August 2017 – Accepted 16 January 2018) Copyright © 2018 Araj et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction increasing health costs. This situation is a major Globally, infection with carbapenem resistant (CR) challenge for the treating clinicians and infection Escherichia coli (Ec) and Klebsiella pneumoniae (Kp) control professionals [1-3,7,8]. In Lebanon, similar as well as other CR Enterobacteriaceae (CRE) had been problems and challenges have been encountered since on the rise, often accompanied by high rates of CRE pathogens were introduced in this country several resistance to a wide range of antimicrobials. These years ago [9,10]. As a result, several studies have been pathogens, together with the dwindling antimicrobial conducted to describe the phenotypic and molecular armament, constitute a most concerning contemporary features of CR E. coli and K. pneumoniae in this threat to health in many countries [1-3]. The Center for country [5,11-15]. Disease Control and Prevention in the USA noted an Because of the very limited available options to increase in the percentages of CRE from 1.2% in 2001 treat CR isolates, this study was undertaken to identify to 4.2% in 2011, where Klebsiella spp. accounted for the types and percentages of antimicrobial agents active the highest increase in proportion, a rise from 1.6% to against CR E. coli and K. pneumoniae resistant to 10.4% [4]. In Lebanon, a notable increase was observed ertapenem, imipenem, and/or meropenem. at our medical center in the percentages of CR, from 2010 to 2016 for Ec (0.1% to 5%) and Kp (0.7% to 8%), Methodology as well as in other hospitals [5,6]. Bacterial Isolates The ominous spread of these pathogens together Consecutive non-duplicate isolates of carbapenem with the scarcity of treatment options results in a serious resistant E. coli (300 isolates) and K. pneumoniae (232 health impact, seen in high rates of morbidity and isolates) at the Clinical Microbiology Laboratory, mortality (up to 75% of infected patients) and Department of Pathology and Laboratory Medicine, Araj et al. – Active agents against CRE J Infect Dev Ctries 2018; 12(3):164-170. American University of Beirut Medical Center Bank # 0346, specified colistin MIC of 4 µg/mL)] were (AUBMC) during the period March 2008 to June 2016 also used to ensure the quality of testing. were investigated. Identification of the isolates was based on standard biochemical methods [5]. Statistical Analysis The source distribution of these E. coli and K. Our data was analyzed using Stata v. 13 software pneumoniae isolates was: urine (39% and 32%), body Package (Stata Corp LP, College Station, Texas, USA) screen (19% and 23%), wound/pus/abscess (17% and for determining the p value, considering less than ≤ 0.05 18%), blood (10% and 9%), respiratory (9% and 5%), as significant. and other fluids (6% and 13%). Results Antimicrobial Susceptibility Testing The MICs (µg/mL) for ertapenem, imipenem and The minimal inhibitory concentrations (MICs) of meropenem against the CR E. coli (n = 300) and K. ertapenem, imipenem and meropenem were determined pneumoniae (n = 232) isolates were 8 and 24, 2 and 4 using the Etest methodology (AB BIODISK, Solna, and 1 and 4, respectively. The MICs (µg/mL) for these Sweden) according to the manufacturer’s guidelines. carbapenems were ≥ 32 ug/mL against both E. coli and The span of MIC levels on these strips ranges between K. pneumoniae isolates. ≤ 0.025 and ≥ 32 µg/mL. The 2016 Clinical and The distribution of the MICs (µg/mL) for Laboratory Standards Institute (CLSI) MICs’ ertapenem, imipenem and meropenem among these CR breakpoints (µg/mL) for Enterobacteriaceae were used E. coli and K. pneumoniae isolates are shown in Figures to interpret the susceptibility category as susceptible, 1 and 2. The MIC (µg/mL) level for the three intermediate and resistant, respectively; for ertapenem: carbapenems ranged between ≤ 0.25 and ≥ 32. The 0.5, 1, 2; for imipenem: 1, 2, 4; for meropenem: 1, 2, 4 [16]. Figure 1. Distribution of carbapenem MICs among E. coli isolates (n = 300). The disk diffusion (DD) antimicrobial susceptibility testing (except for colistin) was done and interpreted according to the CLSI standards [16]. Commercial disks (BBL, Becton Dickinson, USA) were used, with antimicrobial concentrations of: amikacin 30 g, gentamicin 10 g, ciprofloxacin 5 g, trimethoprim/sulfamethoxazole 1.25/23.75 g, tigecyline 15 g, colistin 10 g, fosfomycin/trometamal (200 μg), and aztreonam 30 g. Colistin disk diffusion was used and interpreted according to the study of Gelani et al. [17], where the susceptible, intermediate and resistant zone of inhibition were ≤ 11 mm, 12-13 mm, and ≥ 14 mm, respectively. For screening of carbapenemase-producers, Figure 2. Distribution of carbapenem MICs among K. ertapenem disks (10 g) were used. Those isolates pneumoniae isolates (n = 232). showing < 25 mm zone of inhibition were then tested by Etest to confirm the MICs of ertapenem, imipenem and meropenem. Quality Control The American Type Culture Collection (ATCC) quality control strains of E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were used to ensure correct performance of the Etest and disk diffusion methods. When received in February 2016, the quality control reference strains for colistin [CDC E. coli reference strains MCR-1 (AR-Bank # 0349, specified colistin MIC of 2-4 µg/mL) and MCR-1 (AR- 165 Araj et al. – Active agents against CRE J Infect Dev Ctries 2018; 12(3):164-170. figures reflect the distribution of the MICs up to ≥ 12 K. pneumoniae, regardless of the MICs levels of µg/mL and not up to ≥ 32 µg/mL due to space ertapenem, imipenem and meropenem (Table 1). limitation. The distribution of the CR E. coli and K. Discussion pneumoniae isolates according to their overall The clinical microbiology laboratory (CML) has a carbapenems MICs, as well as carbapenems MICs of ≤ key role in promptly detecting and reporting multi-drug 8 or ≥ 8 µg/mL in relation to their susceptible rates to resistant (MDR) pathogens to guide targeted other antimicrobial agents, namely: amikacin, antimicrobial therapy, which will influence outcome. In gentamicin, ciprofloxacin, trimethoprim- this context, the CML at AUBMC reported the first alert sulfamethoxazole, tigecycline, colistin, fosfomycin and about CRE in Lebanon, when the first imipenem aztreonam are presented in Table1. resistant K. pneumoniae that harbors the bla-OXA-48 Three of the antimicrobial agents tested, gene was recovered from the urine of an 8-year- old girl ciprofloxacin, trimethoprim-sulfamethoxazole and in 2008 [9]. aztreonam, showed very low susceptibility rates (≤ Two years later (July 2010), three Iraqi patients 33%) regardless of the MICs level against both E. coli presented at our medical center seeking treatment. CR and K. pneumoniae isolates, and with little difference E. coli and K. pneumoniae were isolated from different among the three carbapenems. Among specimens of these patients, and the isolates harbored aminoglycosides, the susceptibility rates of gentamicin not only the bla-OXA-48 gene but also the very were low, ranging between 44% and 54%, for both E. threatening bla-NDM-1 gene, producing the novel New coli and K. pneumoniae isolates, and with little Delhi Metallo ß- lactamase (MBL) gene [10]. difference among the 3 carbapenems. On the other Subsequently, the incidence of CR Enterobacteriaceae hand, the E. coli isolates showed higher susceptibility continued to rise [5]. Such critically resistant pathogens rates to amikacin (range: 81% to 97%), compared to the warranted publishing an overview to inform the K. pneumoniae isolates (range: 55% and 86%), as noted medical and paramedical community on the local and for the three carbapenems and regardless of the MICs regional epidemiology of carbapenem resistance, the category. Tigecycline, colistin and fosfomycin showed mechanisms involved, screening and detection high activity (66%-100%) against both CR E. coli and methods, as well as their treatment and control [11]. Table 1. Distribution of CR E. coli (n = 300) and K. pneumoniae (232) isolates according to carbapenem MICs in relation to susceptibility rates (%) of eight other antimicrobial agents. Carbapenems & Percent (%) of susceptible isolates to antimicrobial agents CRE Ħ Ħ Ħ MICs (µg/mL) AMK GENT CIP SXT TIGE COLI FOSFO AZT Ertapenem ≤ 8 E. coli 97* 53 28 16 100 82 89 20* > 8 E. coli 88 47 11 16 99 86 93 10 All E. coli 89 51 19 16 99 84 89 15 ≤ 8 K. pneumoniae 84* 40 33 27 87 94* 86* 30* > 8 K. pneumoniae 68 49 24 21 92 79 67 23 All K. pneumoniae 73 47 15 23 90 82 73 25 Imipenem ≤ 8 E. coli 94* 48 19 14 100 89* 94* 15 > 8 E. coli 85 54 16 23 98 74 87 16 All E. coli 92 53 19 17 99 84 89 15 ≤ 8 K. pneumoniae 86* 44 29 24 89 94* 83* 25 > 8 K. pneumoniae 57 49 21 21 94 73 61 24 All K. pneumoniae 77 48 26 23 93 83 68 25 Meropenem ≤ 8 E. coli 94* 49 21* 17 100 85 92* 15 > 8 E. coli 81 52 8 12 98 82 86 13 All E. coli 92 51 19 15 99 83 91 14 ≤ 8 K. pneumoniae 86* 45 31 25 89 88* 76* 30 > 8 K. pneumoniae 55 51 16 19 94 76 61 23 All K. pneumoniae 73 47 26 24 92 84 66 25 CR: Carbapenem resistant; AMK: amikacin; GEN: gentamicin; CIP: ciprofloxacin; SXT: trimethoprim-sulfamethoxazole; TIGe: tigecycline; COLI: colistin; FOSFO: fosfomycin; AZT: aztreonam; *p value significance ≤ 0.05 determined between MICs ≤ 8µg/mL and > 8µg/mL; The number of E. coli and K. pneumoniae isolates tested, respectively, were: tigecycline (198 and 160 isolates), colistin (156 and 125 isolates), fosfomycin (124 and 68 isolates). 166 Araj et al. – Active agents against CRE J Infect Dev Ctries 2018; 12(3):164-170. Generally, physicians rely on the in vitro carbapenemase (KPC) and OXA-48 -producers in K. antimicrobial susceptibility results of pathogens to pneumoniae isolates remain susceptible to gentamicin, provide optimum therapy to their patients. Empirical while this is rare for New Delhi metallo beta-lactamase combination therapy for CRE infected patients is (NDM) - producers. An in vitro synergistic effect was usually based on the local resistance epidemiology. observed when netilmicin and fosfomycin were tested Definitive therapy, however, should be guided by against a wide range of resistant pathogens. Also, a determination of the in vitro antimicrobial susceptibility combination of carbapenems and aminoglycosides profiles especially when considering combined showed in vitro and in vivo (in animals) synergistic treatment for CRE pathogens. This is very important effects against some KPC-producing K. pneumoniae since only very few drugs remain as last-resort agents isolates, but studies with carbapenemases other than (e.g. colistin, fosfomycin, tigecycline, and KPC are lacking [18,27]. A lack of effect was noted aminoglycosides) [18-22]. when a combination of gentamicin and fosfomycin was In our study, the in vitro activity of these last-resort tested against KPC-2-producing K. pneumoniae. In our agents against the CR E. coli and K. pneumoniae current study, the overall higher activity of amikacin isolates detected at our institution are the core of the compared to that of gentamicin against both CR Ec and following discussion. These results will be considered Kp may be attributed to the type of carbapenemase gene especially in relation to updates reported in the recent involved. For example, in the clinical setting, a study scientific literature [18,19]. from Greece reported a favorable outcome in five Colistin has significant activity against various patients infected with KPC-2-producing K. pneumoniae carbapenemase-producing isolates and is often used in who were treated with aminoglycoside plus colistin combination therapy (e.g., with aminoglycosides, (plus tigecycline in 2 patients) [29]. aztreonam, carbapenems, rifampin, tigecycline, or Tigecycline has also been recommended as part of fosfomycin) with a high (82.1%) clinical cure rate the initial combination regimen in the treatment of [18,23,24]. Caution in its use is advised, to avoid patients with CRE infections in different body sites, development of colistin-resistant Ec & Kp. In our study, other than UTI, especially when tigecycline MIC is ≤ 1 the overall high susceptibility rates (82%-84 % ) of both mg/L (the typical MICs was 0.5 mg/L for E. coli and Ec and Kp to colistin makes it an important first option 1–2 mg/L for K. pneumoniae). The overall crude to use in the treatment of such CR infections (Table 1). mortality when treating with tigecycline was higher Fosfomycin has also been considered as part of a with monotherapy than with combined treatment (40– combination regimen (including at least one more 80% vs 0–33%) [18]. In our study, tigecycline showed active agent, e.g. with tigecycline and colistin) in the very high in vitro activity against both CR Ec and Kp, treatment of invasive infections with CRE pathogens making it an excellent agent in combined treatment [25]. Its use in combinations, however, resulted in options for such CR pathogens. Low in vitro resistance varied synergistic activity against CRE depending on rates against tigecycline were also reported in a 2008 the type of pathogen and the antibiotics used [26]. For study from our institution, in MDR and ESBL Ec (0%) example, Samonis et al. [27] reported that the and Kp (3%) [29]. The very low rates noted in the synergistic effect of fosfomycin was 55% to 79% with previous and current studies indicate a sustainable carbapenems, 7.1% to 36% with colistin, 21% to 30% activity of tigecycline against resistant isolates at our with tigecycline, and 25% to 43% with gentamicin. In center. this study, fosfomycin showed high activity (89-91%) Aztreonam testing was included in this study against the CR Ec, while the susceptibility of CR Kp because MBLs can hydrolyze carbapenems and all the was lower (66-73%). We conclude that fosfomycin can available beta-lactams with the exception of aztreonam. remain a viable option for the treatment of CR Whether aztreonam remains an option for treating pathogens especially Ec (Table 1). These levels are infections due to MBL producers that test susceptible to close to those we reported in 2012 on the fosfomycin this agent remains to be determined; currently there is susceptibility of ESBL-producing pathogens, where its no clinical experience with aztreonam for the treatment activity was higher against ESBL-producing E. coli of invasive infections due to CRE [30]. In our study, isolates (86%) than against K. pneumoniae isolates however, a very low aztreonam susceptibility (14-25%) (62%) [28]. was shown by both CR Ec and CR Kp isolates, Aminoglycosides have been used in combination indicating that the majority of these isolates were not with other classes of antibiotics with different results MBL producers. An earlier study on the CR genes [18,27]. For example, some K. pneumoniae involved in K. pneumoniae and E. coli isolates from our 167 Araj et al. – Active agents against CRE J Infect Dev Ctries 2018; 12(3):164-170. center indicated the prevalence of bla-OXA-1, bla- In our study the carbapenem rates of MIC ≤ 8 mg/L CTXM-15, bla-TEM-1, bla-CMY-2, bla-OXA-48, and for ertapenem, imipenem and meropenem were higher porin channel genes, and few had the NDM-1 gene [12]. when tested against E. coli: 48%, 74% and 82%, The antimicrobial agents discussed above have respectively, than against K. pneumoniae: 27%, 58% been considered for possible clinical relevance to treat and 63%, respectively. Such rates, especially for patients infected with CRE. The question of the imipenem and meropenem, in combination with the superior effectiveness of mono- or combined other in vitro active agents, can provide an adequate antimicrobial treatment of patients infected with CRE option in treating patients, particularly those with CR E. pathogens was excellently reviewed by Rodríguez- coli infection. Baño and colleagues in Spain and Karaiskos and Studies at our medical center since 2008 have been Giamarellou in Greece, looking at microbiological addressing molecular characterizations, resistance diagnosis and treatment of infections with mechanisms, genes involved, and other aspects of CR carbapenemase-producing Enterobacteriaceae [18,19]. Ec and Kp isolates [9,10,12-14]. The first CRE detected These comprehensive studies suggest a more favorable in Lebanon was a K. pneumoniae recovered from a 7- outcome (e.g. reduced mortality) using a combined year-old female child, which was characterized by PCR antimicrobial treatment for patients infected with CRE experiments using primers for multiple β-lactamase and pathogens. Besides, combined therapy may maximize carbapenemase genes: TEM, SHV, CTX-M, GES, bacterial killing (synergistic effect) and minimize KPC, IMI, OXA-1 ⁄ 2 ⁄ OXA-10 ⁄ 18, OXA-23 ⁄ 58, IMP, bacterial resistance. For example, Tumbarello et al. VIM, SPM, GIM and SIM. The only positive result was 2012 [31] reported a higher mortality rate among for a blaOXA-48-like gene [9]. Interestingly, this was patients treated with monotherapy compared to only the fourth report about this gene worldwide. combined therapy (54.3% vs 34.1%); the combination Subsequently, increasing numbers of CRE have been included tigecycline, colistin, and meropenem. noticed; some harbored the very threatening bla NDM- In addition, the inclusion of a carbapenem in the 1 gene, detected for the first time in Lebanon, in E. coli combined treatment showed promising results. For and K. pneumoniae isolates recovered from Iraqi example, Qureshi et al., reported lower mortality when patients coming for medical care at AUBMC [10]. carbapenem was used in combination with colistin or The underlying mechanisms and genes responsible tigecycline, compared to a combination that lacked a for the carbapenem-resistance in K. pneumoniae and E. carbapenem (12.5% vs 66.7%) [32]. Moreover, the coli revealed the presence of different Beta-lactamase effect of carbapenems in the combination was gene profiles including: bla-OXA-1, bla-CTXM-15, noticeable if the carbapenem MIC was ≤ 8 mg/L, and it bla-TEM-1, bla-CMY-2, bla-OXA-48 and NDM-1 was added to one or two in vitro fully active drugs genes in both genera; in addition, the K. pneumoniae (including colistin, tigecycline, an aminoglycoside or isolates were found to lack outer membrane porin fosfomycin) for such infections. Carbapenems, despite (OmpC and OmpF) encoding genes, while E. coli being hydrolyzed by carbapenemases, may retain some harbored these porin genes [12]. In addition, the activity against carbapenemase-producing isolates prevalence of carbapenem resistance encoding genes [18,19,33,34]. Such findings were vividly reflected in and their correlation with corresponding MIC against the study by Daikos et al. [20] who reported a lower ertapenem, imipenem and meropenem were studied mortality rate with treatment by a carbapenem- among CR E. coli (n=76) and CR K. pneumoniae containing combination with another active drug, such (n=54) isolates. The prevalence of blaOXA-48, as an aminoglycoside, colistin or tigecycline, than with blaNDM-1, blaTEM-1 and blaCTX-M-15 among the a combination without a carbapenem (19.3% vs 30.6%). E. coli isolates were 36%, 12%, 20% and 80%, It was important that the carbapenem had a MIC ≤ 8 respectively, while among K. pneumoniae isolates they mg/L [20]. Akova et al. found similar results [35]. were 37%, 28%, 28% and 72%, respectively. The Interestingly, the efficacy of using dual carbapenem presence of more than one carbapenem resistance (i.e., meropenem plus ertapenem) therapy has been encoding gene and/ or ESBL encoding gene did not shown in animal model of infection (i.e., mouse have an effect on the MIC value in K. pneumoniae pneumonia) and in humans infected with KPC isolates, while in E. coli they resulted in higher MIC producers. The suggested explanation was that values [14]. ertapenem most likely acts as a “suicide” molecule for We also assessed the effects of antimicrobial carbapenemase activity, whereas the more active drug, combination therapy against bacteria with different CR meropenem, retains its efficacy [36]. genes using BALB/c mice [13]. The mice were injected 168 Araj et al. – Active agents against CRE J Infect Dev Ctries 2018; 12(3):164-170. 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Clin Microbiol American University of Beirut Medical Center Infect 11: 115–121. PO Box 11-0236, Beirut, Lebanon 1107-2020 25. Pontikis K, Karaiskos I, Bastani S, Dimopoulos G, Kalogirou Phone: +(961) -1- 350 000 ext 5215 M, Katsiari M, Oikonomou A, Poulakou G, Roilides E, Fax: +(961) -1- 370 845 Giamarellou H (2014) Outcomes of critically ill intensive care Email: garaj@aub.edu.lb unit patients treated with fosfomycin for infections due to pandrug-resistant and extensively drug resistant Conflict of interests: No conflict of interests is declared. carbapenemase-producing Gram-negative bacteria. Int J Antimicrob Agents 43: 52–59. 26. Falagas ME, Kastoris AC, Kapaskelis AM, Karagoergopoulos DE (2010) Fosfomycin for the treatment of multidrug-resistant,

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The Journal of Infection in Developing CountriesUnpaywall

Published: Mar 31, 2018

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