ANTIMICROBIAL SUSCEPTIBILITY - EMERGENCE OF CARBAPENEMASE-PRODUCING E. COLI

Transcription

1 RESISTANCE? Sample ES-03 (2018) was a simulated urinary tract culture from a 50-year-old male in the ICU on a ventilator. Additional clinical details are a history of travel to the United Kingdom, Pakistan, and Hong Kong in the last 12 months. This organism harbored several β-lactamases including NDM-1 (New Delhi Metallo-beta-lactamase) among other mechanisms of resistance, and was sent to API participants as an ungraded educational challenge to determine the ability of contemporarily used antimicrobial susceptibility testing (AST) products/methods 1-6 to recognize and appropriately categorize the XDR pattern in this Enterobacteriaceae species. The sample contained an Escherichia coli organism in pure culture having an extensively-drug resistant (XDR) antibiogram profile. This profile reveals very few active agents (Table 1) and a phenotypic resistance pattern that includes: Aminoglycosides (amikacin, gentamicin, tobramycin); Cephalosporins (cefepime, ceftazidime, ceftriaxone); Fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin); Carbapenems (doripenem, ertapenem, imipenem, meropenem); Monobactams (aztreonam); Penicillins (ampicillin, amoxicillin); Tetracyclines (tetracycline); and various β-lactam/β-lactamase inhibitor combinations such as those with clavulanic acid, sulbactam, avibactam, tazobactam and vaborbactam. Organism identification responses (880 overall) were acceptable for 99.9% of responding laboratories; excellent performance! The five most commonly selected responses were: E. coli (717, 81.1%); E. coli, ESBL strain (102, 11.5%); E. coli, possible CRE - would refer (25, 2.8%); Gram-negative organisms (12, 1.4%); and E. coli, possible ESBL - would refer (11, 1.2%). The remaining seven responses were for E. coli and variations of the ESBL or CRE or referral identifications. Only one error (Enterobacter cloacae from a MicroScan system) was not E. coli. Organism Identification, Features and Taxonomy Escherichia coli are part of the normal intestinal flora in nearly all healthy humans. They are Gramnegative, oxidase-negative, rod shaped, facultatively anaerobic members of the Enterobacteriaceae. 7 E. coli are widely distributed in nature and have been observed in nearly every anatomical clinical culture specimen in humans including urine (where they are the most frequently encountered pathogen), sputum, wound, skin and subcutaneous tissue, blood, and other normally sterile body fluids. 7,8 The correct identification of E. coli can be easily achieved using standard biochemical algorithms and tests or by the use of automated systems including BD Phoenix, MicroScan, Vitek, and Matrix-Assisted Laser Desorption/Ionization Time Of Flight (MALDI-TOF) mass spectrometry E. coli grow well on general and on most selective/differential agar media designed for primary isolation of Gram-negative bacilli. After overnight incubation at 35 C, colony morphology will most often appear as large, round, entire, gray to white colonies which are often hemolytic on blood containing agars, but can be American Proficiency Institute rd Test Event 1

2 Table 1. List of expected antimicrobial susceptibility testing MIC and categorical interpretation results from reference broth microdilution methods for the E. coli strain found in ES-03 (2018). CLSI/US-FDA breakpoints were applied where appropriate. Antimicrobial agents listed by CLSI susceptibility category (MIC in µg/ml): a Susceptible Intermediate Resistant Colistin (0.25) Chloramphenicol (16) b Amikacin (>32) Nitrofurantoin (16) Amoxicillin-Clavulanate (>32/16) Tigecycline (0.5) b Ampicillin (>64) TMP/SMX (0.12/2.28) c Ampicillin-Sulbactam (>64/32) Aztreonam (>16) Cefazolin (>16) Cefepime (>16) Cefpodoxime (>64) Ceftaroline (>32) Ceftazidime (>32) Ceftazidime-Avibactam (>32/4) Ceftolozane-Tazobactam (>32/4) Ceftriaxone (>8) Cefuroxime (>64) Ciprofloxacin (>4) Delafloxacin (8) Doripenem (>8) Ertapenem (>2) Gentamicin (>16) Imipenem (8) Levofloxacin (>16) Meropenem (32) Meropenem-Vaborbactam (32/4) Piperacillin-Tazobactam (>128/64) Plazomicin (>128) Tetracycline (>16) Tobramycin (>16) a. Fosfomycin, Trimethoprim alone, and Sulfisoxazole were also active. b. Not appropriate for testing and reporting on a UTI specimen. c. TMP/SMX = Trimethoprim/Sulfamethoxazole non-hemolytic. This species displays lactose fermentation and grows well on MacConkey agar, a characteristic shared with other Enterobacteriaceae. 7 E. coli also commonly produce a green metallic sheen when grown on Eosin Methylene Blue (EMB) agar. Production of indole from tryptophan is American Proficiency Institute rd Test Event 2

3 observed with E. coli as well as K. oxytoca, but other colonial morphology characteristics usually differentiate these two species. 7 Most E. coli biotypes are motile by peritrichous flagella and ferment d-glucose, which differentiates E. coli from Shigella spp., a phenotypically similar genus with nearly the same DNA-DNA hybridization and whole genome sequence analysis when compared to E. coli. 7 Antimicrobial Susceptibility Testing (Ungraded) Participants were instructed to perform antimicrobial susceptibility testing (AST) on this less commonly isolated resistant variant of E. coli. The reference laboratory AST was conducted in triplicate by CLSI/ISO broth microdilution method, 1 and susceptibility categorical interpretations were assigned based on CLSI document M100Ed28E (2018), where available. 3 The reference laboratory reported MIC results for 32 agents (Table 1). Results for 24 compounds or drug combinations were received from participants in sufficient numbers ( 96 responses) to allow comment on AST practices and to determine accuracy (Table 2). Consensus grading criteria ( 80% categorical agreement among reporting laboratories by method [disk diffusion and MIC]) are presented in Table 2. Among the tabulated results, dominantly acceptable test accuracy was observed, although grading was not applied to participant results. Table 1 recognizes only four active antimicrobials (colistin, nitrofurantoin, tigecycline, and trimethoprim / sulfamethoxazole [TMP/SMX]) when tested against the XDR E. coli. Three additional active drugs were identified by consensus participant results performed at small numbers of sites. These agents were fosfomycin (5 participant results), trimethoprim (39), and sulfisoxazole (1). Polymyxin B would also be inferred as susceptible from the colistin result. 3-5 Four test methods or products (DD, BD Phoenix, MicroScan, and Vitek 2) contributed accurate susceptible test results (47 total results) for these additional active drugs. Table 2 lists nitrofurantoin, tigecycline, and TMP/SMX results (1,668 results) as the most commonly tested active agents, some UTI-directed. The accuracy across the AST results for active agents ranged from 94.6% (trimethoprim MIC tests) to 100% (multiple other active drugs and methods). Note that tigecycline does not have a clinical induction for UTI. Several other antimicrobials were inappropriately tested and reported by small numbers of laboratories (azithromycin, clarithromycin, erythromycin, linezolid, and vancomycin) for this pathogen and UTI infection type. Furthermore, 229 results from appropriate but inactive antimicrobials were tested by a small number of laboratories, and were not tabulated in Table 2. Among these drugs only four (4.7%) false-susceptible results were identified, for doripenem (2 of 11; 18.2%) and norfloxacin (2 of 20; 10.0%). Drugs recently approved by the FDA to be focused on unmet medical needs (MDR and XDR Gramnegative pathogens like this sample) were also tested by a few participants. The lack of activity against this metallo-beta-lactamase (MBL)- producing isolate was documented for ceftazidime-avibactam, American Proficiency Institute rd Test Event 3

4 ceftolozane-tazobactam, meropenem-vaborbactam and plazomicin (16 results total from MicroScan and Vitek 2 products); see Table 1. Table 2. Participant AST performance for 24 selected agents tested by disk agar diffusion (DD) and/or quantitative MIC methods when applied to the E. coli ES-03 (2018). Only agents with approximately 100 or more responses for either or both test methods were tabulated. Satisfactory DD MIC Antimicrobial susceptible category No. % No. % Amikacin Resistant Amoxicillin-Clavulanate Resistant Ampicillin Resistant Ampicillin-Sulbactam Resistant Aztreonam Resistant Cefazolin Resistant Cefepime Resistant Cefotaxime Resistant Cefoxitin Resistant Ceftazidime Resistant Ceftriaxone Resistant Cefuroxime Resistant Ciprofloxacin Resistant Ertapenem Resistant Gentamicin Resistant Imipenem Resistant Levofloxacin Resistant Meropenem Resistant Nitrofurantoin Susceptible Piperacillin-Tazobactam Resistant Tetracycline Resistant Tigecycline b Susceptible b b Tobramycin Resistant TMP/SMX c Susceptible a. Susceptibility categories determined by CLSI M100-S28 (2018), where available. US-FDA criteria was used for tigecycline. Percentage of categorical responses considered correct by consensus among participant results and/or reference laboratory values; see Table 1. b. Not appropriate for testing and reporting on a UTI specimen. c. TMP/SMX = Trimethoprim-Sulfamethoxazole. Table 2 lists DD method categorical results from approximately 25 laboratories (21 drugs; 185 categorical results). For this AST method, only four results were not correct (2.2%) or 97.8% accuracy. The MIC test American Proficiency Institute rd Test Event 4

5 systems were similarly acceptable with percentage accuracy rates ranging from 87.7% (imipenem) to 100.0% (11 drugs). The most compromised AST performance was the commercial MIC AST detection of carbapenem resistance where false-susceptible and -intermediate results (99 of 1,304 carbapenem results) were observed for doripenem (18.2% of results) > imipenem (12.3%) > meropenem (8.5%) > ertapenem (0.3%). The flaw in the identification of the NDM-1 MBL was greatest for MicroScan MIC systems where 96 of 99 (97.0%) of reported errors came from that product. Emerging Resistance and Molecular Epidemiology Among E. coli Isolates The challenge isolate (ES ) was resistant to all β-lactams, including carbapenems, newer β-lactam/β-lactamase inhibitor combinations such as ceftazidime-avibactam and meropenemvaborbactam, fluoroquinolones, aminoglycosides, and tetracyclines. WGS analysis of this isolate revealed the presence of five genes encoding β-lactamases (Table 3), including the MBL NDM-1. Other β-lactamases present were: (1) the extended-spectrum enzyme (ESBL) CTX-M-15; (2) two transferrable (plasmidic) AmpC genes, CMY-4 and DHA-1; and (3) OXA-1. The latter, also known as OXA-30, hydrolyzes cefepime 13 and is usually carried along with CTX-M Table 3. Resistance genes detected by WGS in the challenge isolate ES-03 (2018). Antimicrobial class affected Aminoglycosides β-lactams Cephalosporins Carbapenems Fluoroquinolones Sulfonamides Tetracyclines Macrolides and streptogramin B Resistance gene detected arma, aac(3)-iia, aac(6`)-ib-cr blactx-m-15, blaoxa-1, blacmy-4, bladha-1 blandm-1 aac(6`)-ib-cr sul1 tet(a) mph(a), mph(e), msr(e) NDM-1 was initially reported from Klebsiella pneumoniae and E. coli strains from India. 15,16 Among MBL enzymes, NDM-1 and its 20 described variants have demonstrated a remarkable ability to disseminate. These genes have rapidly spread worldwide among many Gram-negative bacterial species. 16 According to the Centers for Disease Control and Prevention (CDC), a total of 379 isolates producing NDM enzymes were observed in the United States (U.S.) during The number of isolates per state ranged from 0 American Proficiency Institute rd Test Event 5

6 (17 states) to 109 (Illinois) isolates in Other states with an elevated number of NDM-producing isolates were California (75 isolates), Washington (31 isolates), and Colorado (25 isolates). Carbapenem-resistant E. coli isolates and isolates carrying the gene encoding NDM enzymes are included in the National Notifiable Diseases Surveillance System (NNDSS) and should be reported to the CDC ( when isolated. Noteworthy, MBLs confer resistance to all β-lactams with the exception of monobactams, 18 represented in the susceptibility results by aztreonam; however, due to the presence of other β-lactamases, this isolate was also resistant to aztreonam. Another important characteristic of MBLs is the lack of inhibition by clinically available older generation β-lactamase inhibitors such as clavulanate, sulbactam, or tazobactam, and the newer inhibitors, avibactam and vaborbactam (Table 4). There are many initiatives to develop MBL inhibitors, but there are currently no such agents in the late stage of clinical development. Table 4. Inhibition of β-lactamases by older and new β-lactamase inhibitors. β-lactamase Clavulanate Tazobactam Avibactam Vaborbactam TEM or SHV with ESBL Class A CTX-M PER, VEB, GES KPC - - Class B IMP, VIM, NDM Class C Chromosomal AmpC - - Plasmidic ACC, DHA, FOX, LAT, MIX, MIR, ACT - - Class D Penicillinase-type (OXA-1, -31, -10, -13) Carbapenemase-type (OXA-23, -40, -48, -58) - - OXA-48 (some isolates) - = inhibitory activity observed in vitro; - = no inhibitory activity in vitro American Proficiency Institute rd Test Event 6

7 In addition to the β-lactamase genes, this isolate carried a gene encoding the 16S rrna methyltransferase arma that codifies resistance to all clinically used aminoglycosides, including the newly approved plazomicin. 19 Isolates carrying 16S rrna methyltransferase genes are uncommon in the U.S., but many isolates carrying blandm harbor 16S rrna methyltransferase genes. This isolate also carried two aminoglycoside modifying enzymes (AMEs): aac(3)-iia that confers resistance to gentamicin, tobramycin, and netilmicin, among others; and aac(6`)-ib-cr that is able to modify amikacin, gentamicin, and fluoroquinolones. 20 Both genes are widely disseminated in U.S. hospitals. 21 Interestingly, this isolate carried sul1 that encodes resistance to sulfonamides, but the MIC values for trimethoprim-sulfamethoxazole were low (MIC, 0.12/2.28 µg/ml). The presence of resistance genes does not always indicate that this gene is being expressed and could occur without a resistant phenotype. This isolate also harbored tet(a) that confers resistance to tetracyclines, but not to tigecycline. This glycylcycline was designed to be stable against tet(a) ribosomal modifications. 22 Other resistance genes detected included mph(a), mph(e), and msr(e) that encode resistance to macrolides and streptogramin B. MDR and XDR E. coli isolates are uncommon when compared to XDR in K. pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, but these isolates can occur, and clinical laboratories should be prepared to test and report alternative agents for the treatment of these organisms. Therapeutic Considerations for UTIs Caused by MDR and XDR E. coli The AST results showed that this E. coli isolate was resistant to nearly all antimicrobial agents tested, including fluoroquinolones, aminoglycosides, broad-spectrum cephalosporins, carbapenems, piperacillintazobactam, and the recently approved β-lactam/β-lactamase inhibitor combinations ceftolozanetazobactam, ceftazidime-avibactam, and meropenem-vaborbactam (Table 1). Interestingly, this XDR NDM-producing organism remained susceptible to some of the oldest antimicrobial agents indicated for treatment of UTIs, such as trimethoprim-sulfamethoxazole (TMP-SMX), nitrofurantoin, fosfomycin, and colistin. Moreover, the isolate was also susceptible to tigecycline, which was initially approved by the US- FDA in 2005 and does not have a UTI indication. 6 TMP-SMX is a combination agent prescribed for multiple clinical indications and is the fourth most commonly prescribed antimicrobial in the U.S. Although TMP-SMX has been effective in the treatment of UTIs caused by TMP-SMX susceptible organisms for more than three decades, sulfamethoxazole can cause patient allergic reactions and trimethoprim is associated with a greater risk of acute kidney injury (AKI) and hyperkalemia compared with other antimicrobial agents for UTI, especially among the elderly population. 23,24 American Proficiency Institute rd Test Event 7

8 Nitrofurantoin was approved by the US-FDA in 1953 and has been used clinically for more than 60 years. Nitrofurantoin is mainly indicated for the treatment of UTIs in females because it does not achieve therapeutic levels in prostate tissue and concomitant prostatitis cannot be ruled out in males. Clinical trials have shown that in the treatment of uncomplicated UTIs nitrofurantoin is equivalent to TMP-SMX, ciprofloxacin, and amoxicillin-clavulanate (all administered for 3 days) when given for 5 or 7 days, but less effective than comparators when administered for only 3 days. Nitrofurantoin achieves therapeutic concentration only in the lower urinary tract, restricting its indication to the treatment of cystitis. Toxicity of short-term ( 14 days) use of nitrofurantoin is generally mild and predominantly gastrointestinal. Indeed, large population-level datasets point to a significant confidence in nitrofurantoin even in patient groups for whom this drug has historically been discouraged, such as in men and the elderly. 25 Fosfomycin is an older broad-spectrum bactericidal agent that inhibits peptidoglycan formation, and it has a broad spectrum of activity against a wide range of Gram-positive and Gram-negative bacteria, including ESBL- and MBL-producing strains. 26,27 Thus, oral fosfomycin has been increasingly used as an alternative or even as a first-line treatment of UTI because of the increased incidence of UTIs caused by MDR organisms. Although clinical studies have demonstrated the efficacy of fosfomycin in the treatment of lower UTI caused by MDR E. coli, only 70 85% of the treatments with fosfomycin result in clinical success. Reasons for treatment failure suggest inadequate urinary drug concentrations and/or a large interindividual pharmacokinetic variation. 28 The polymyxins, including colistin (polymyxin E) and polymyxin B, are used as a last-resort treatment of MDR bacterial infections caused by CRE, MDR-P. aeruginosa, and A. baumannii. Although the polymyxins are active in vitro against some MDR organisms, these compounds have been associated with unsatisfactory clinical efficacy and a wide range of adverse events, especially AKI, and should be avoided when other options are available Lastly, tigecycline is a glycylcycline agent and a derivative of minocycline. Tigecycline is not recommended for the treatment of UTIs due to low peak serum concentrations and limited excretion into urine. In the absence of effective and safe antimicrobial options, high-dose tigecycline (e.g., loading dose of mg followed by mg daily) can be considered as an alternative option to treat UTIs due to MDR organisms; however, higher frequency of adverse effects has been reported for tigecycline in comparison to other agents. Moreover, tigecycline monotherapy is not recommended for the treatment of moderate-to-severe UTIs, particularly in the presence of bacteremia and should be combined with other active agents. 32,33 American Proficiency Institute rd Test Event 8

9 In summary, this case is a good example of an XDR organism that can potentially be treated with old, well-established antimicrobial agents. This emphasizes the growing importance of hospital-based antimicrobial stewardship. 34 References 1. CLSI. M07Ed11E. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard: eleventh edition. Wayne, PA: Clinical and Laboratory Standards Institute CLSI. M02Ed13E. Performance standards for antimicrobal disk susceptibility tests; Thirteenth Edition. Wayne, PA: Clinical and Laboratory Standards Institute CLSI. M100Ed28E. Performance standards for antimicrobial susceptibility testing: 28th informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute EUCAST (2018). Breakpoint tables for interpretation of MIC's and zone diameters. Version 8.0, January Available at t_tables.pdf. January USCAST. Breakpoint tables for interpretations of MICs and Zone Diameters. Version 4.0, July Tygacil. Tygacil Package Insert. Philadelphia, PA: Pfizer, Inc., Available at Date Accessed October 15, Jorgensen JH, Pfaller MA, Carroll KC, et al. Manual of Clinical Microbiology, 11th ed. Washington, D.C.: ASM Press, Leber AL, editor. Clinical Microbiology Procedures Handbook, 4th Ed. Washington, D.C.: ASM Press, O'Hara CM, Miller JM. Evaluation of the MicroScan rapid neg ID3 panel for identification of Enterobacteriaceae and some common gram-negative nonfermenters. J Clin Microbiol. 2000; 38: Abbott SL, O'Connor J, Robin T, Zimmer BL, Janda JM. Biochemical properties of a newly described Escherichia species, Escherichia albertii. J Clin Microbiol. 2003; 41: American Proficiency Institute rd Test Event 9

10 11. He Y, Li H, Lu X, Stratton CW, Tang YW. Mass spectrometry biotyper system identifies enteric bacterial pathogens directly from colonies grown on selective stool culture media. J Clin Microbiol. 2010; 48: Clark AE, Kaleta EJ, Arora A, Wolk DM. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clin Microbiol Rev. 2013; 26: Aubert D, Poirel L, Chevalier J, Leotard S, Pages JM, Nordmann P. Oxacillinase-mediated resistance to cefepime and susceptibility to ceftazidime in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2001; 45: Castanheira M, Farrell SE, Deshpande LM, Mendes RE, Jones RN. Prevalence of beta-lactamaseencoding genes among Enterobacteriaceae bacteremia isolates collected in 26 U.S. hospitals: report from the SENTRY Antimicrobial Surveillance Program (2010). Antimicrob Agents Chemother. 2013; 57: Yong D, Toleman MA, Giske CG, et al. Characterization of a new metallo-beta-lactamase gene, blandm-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother. 2009; 53: Nordmann P, Poirel L, Walsh TR, Livermore DM. The emerging NDM carbapenemases. Trends Microbiol. 2011; 19: Centers for Disease Control and Prevention. Patients with NDM-producing Carbapenem-resistant Enterobacteriaceae (CRE) reported to the Centers for Disease Control and Prevention (CDC) as of December 2017, by state. Tracking CRE Web site. Updated February 27, Accessed October 29, Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo-beta-lactamases: the quiet before the storm? Clin Microbiol Rev. 2005; 18: Castanheira M, Davis AP, Mendes RE, Serio AW, Krause KM, Flamm RK. In vitro activity of plazomicin against Gram-negative and Gram-positive isolates collected from U. S. hospitals and comparative activity of aminoglycosides against carbapenem-resistant Enterobacteriaceae and isolates carrying carbapenemase genes. Antimicrob Agents Chemother. 2018; 62:e Ramirez MS, Tolmasky ME. Aminoglycoside modifying enzymes. Drug Resist Updat. 2010; 13: Castanheira M, Davis AP, Serio AW, Krause KM, Mendes RE. In vitro activity of plazomicin against Enterobacteriaceae isolates carrying genes encoding aminoglycoside-modifying enzymes most common in United States cencus difivisions. Diag Microbio Infect Dis. 2018:accepted. American Proficiency Institute rd Test Event 10

11 22. Bradford PA, Petersen PJ, Tuckman M, Jones CH. In vitro activity of tigecycline and occurrence of tetracycline resistance determinants in isolates from patients enrolled in phase 3 clinical trials for community-acquired pneumonia. Clin Microbiol Infect. 2008; 14: Crellin E, Mansfield KE, Leyrat C, et al. Trimethoprim use for urinary tract infection and risk of adverse outcomes in older patients: cohort study. Bmj. 2018; 360:k Ho JM, Juurlink DN. Considerations when prescribing trimethoprim-sulfamethoxazole. CMAJ. 2011; 183: Huttner A, Verhaegh EM, Harbarth S, Muller AE, Theuretzbacher U, Mouton JW. Nitrofurantoin revisited: a systematic review and meta-analysis of controlled trials. J Antimicrob Chemother. 2015; 70: Flamm RK, Rhomberg PR, Watters AA, Sweeney K, Ellis-Grosse EJ, Shortridge D. Activity of fosfomycin when tested against US contemporary bacterial isolates. Diagn Microbiol Infect Dis. 2018:in press. 27. Patwardhan V, Singh S. Fosfomycin for the treatment of drug-resistant urinary tract infections: potential of an old drug not explored fully. Int Urol Nephrol. 2017; 49: Falagas ME, Vouloumanou EK, Togias AG, et al. Fosfomycin versus other antibiotics for the treatment of cystitis: a meta-analysis of randomized controlled trials. J Antimicrob Chemother. 2010; 65: Baradaran S, Black DJ, Keyloun KR, Hansen RN, Gillard PJ, Devine B. The impact of acute kidney injury on the risk of mortality and health care utilization among patients treated with polymyxins for severe Gram-negative infections. Open Forum Infect Dis. 2018; 5:ofy Paul M, Daikos GL, Durante-Mangoni E, et al. Colistin alone versus colistin plus meropenem for treatment of severe infections caused by carbapenem-resistant Gram-negative bacteria: an openlabel, randomised controlled trial. Lancet Infect Dis. 2018; 18: Vardakas KZ, Mavroudis AD, Georgiou M, Falagas ME. Intravenous colistin combination antimicrobial treatment vs. monotherapy: a systematic review and meta-analysis. Int J Antimicrob Agents. 2018; 51: Tasina E, Haidich AB, Kokkali S, Arvanitidou M. Efficacy and safety of tigecycline for the treatment of infectious diseases: a meta-analysis. Lancet Infect Dis. 2011; 11: Kaewpoowat Q, Ostrosky-Zeichner L. Tigecycline : a critical safety review. Expert Opin Drug Saf. 2015; 14: American Proficiency Institute rd Test Event 11

12 34. Wiens J, Snyder GM, Finlayson S, Mahoney MV, Celi LA. Potential adverse effects of broadspectrum antimicrobial exposure in the intensive care unit. Open Forum Infect Dis. 2018; 5:ofx270. American Proficiency Institute rd Test Event 12