ANTIMICROBIAL SUSCEPTIBILITY ADDRESSING ACCURATE TESTING OF MULTIDRUG- RESISTANT ACINETOBACTERS

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1 RESISTANT ACINETOBACTERS Sample ES-01 was a simulated blood culture and a duplicate of a strain used in 2009 (ES-03). Participants were instructed to perform organism isolation, identification of the pathogen, and susceptibility testing 1-4 by their routinely utilized method (system). The sample contained a multidrugresistant (MDR) Acinetobacter baumannii strain in a pure culture that expressed a plasmid-borne OXA-23 β-lactamase. This organism was originally isolated from a patient s blood culture in Italy during an antimicrobial resistance surveillance in This specimen was distributed as an educational challenge and grading was not applied to the results. Responses of A. baumannii (613 participants; 74.1%), Acinetobacter sp. (152; 18.4%), Gram negative organism (19; 2.3%), A. baumannii complex (9; 1.1%), and A. baumannii / A. haemolyticus (7; 0.8%) would be considered acceptable identification performance. The satisfactory level of identification was achieved by 800 (96.7%) of participating laboratories. Only 38 facilities recorded unsatisfactory responses, which included Pseudomonas spp., not aeruginosa or Pseudomonas spp. alone (27; 1.7%). The level of pathogen identification to the species level improved significantly (64.0 to 74.1%) since The overall acceptable identification rate also increased from 94.4 to 96.7%, participants well done! Organism Identification Acinetobacter spp. are strictly aerobic Gram negative, non-fastidious, non-fermentative, non-motile, catalase-positive and oxidase-negative coccobacilli. They may be variable upon Gram-staining, and the morphologic characteristics may change depending on the growth phase. 5 This bacterium has a tendency to retain the crystal violet, making destaining difficult or incomplete, and may be misidentified as Gram positive cocci. Acinetobacter spp. grow readily on standard laboratory culture media (e.g., MacConkey agar or sheep blood agar) and typically form smooth, sometimes mucoid, white to grayishwhite colonies. For this clinical isolate, the optimum growth temperature is C or higher, but other genomic species require lower temperatures. The genus Acinetobacter (DNA G+C content 39-47%) was initially classified in the family Neisseriaceae, but is now classified in the Moraxellaceae family. 6 According to NCBI, greater than 30 species within the Acinetobacter genus have been identified. 7 Four closely related species, A. calcoaceticus, A. baumannii, Acinetobacter pittii (formerly genomospecies 3) and Acinetobacter nosocomialis (formerly genomospecies 13TU) are difficult to distinguish based on phenotypic properties, and therefore referred to as the A. calcoaceticus - A. baumannii complex. The use of automated identification systems (MicroScan, API 20NE, VITEK, VITEK 2, etc.) to identify Acinetobacter spp. to the species level has been associated with poor accuracy. Most commercial systems are unable to distinguish among the clinically-relevant strains of the A. calcoaceticus - A. baumannii complex, with A. baumannii being the main species associated with human clinical infections. 8 The reference standard method for identification of Acinetobacter spp. is likely DNA-DNA

2 hybridization, but this method and other molecular techniques are not practical for routine use in most clinical laboratories. Alternatively, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a promising rapid, high-throughput molecular method that has been recently shown to have the ability to differentiate among the Acinetobacter taxa, including species within the A. calcoaceticus - A. baumannii complex. 9 Antimicrobial Susceptibility Testing (Ungraded) Participants were requested to perform antimicrobial susceptibility testing on this A. baumannii. This strain was selected to challenge proper identification of an important emerging non-fermentative bacillus, and to determine antimicrobial susceptibility across numerous classes of indicated antimicrobial agents. The initial reference laboratory antimicrobial susceptibility testing was conducted by standardized reference broth microdilution methods 1 and susceptibility was determined based on CLSI document M100-S24 breakpoints. 3 The reference laboratory testing reported a total of eight agents (Table 1) that demonstrated potent antimicrobial activity against this strain (MIC, 4 µg/ml); two drugs were listed as possessing intermediate susceptibility and 21 drugs were noted to have no measurable (off-scale MIC values) activity against this A. baumannii. Consensus grading criteria ( 80% categorical agreement among all participating and reporting laboratories and methods [disk diffusion or DD and MIC]) are listed in Table 2 for the most reported antimicrobials ( 10 responses by one or more methods). The DD method results generally showed accurate and consistent categorization of antimicrobial activity except for trimethoprim / sulfamethoxazole (42.9% correct) and ceftriaxone (intermediate susceptibility at 81.8%; see discussion below). The overall rate of correctly categorized DD responses was 91.0%. For the MIC methods, these commercial systems had accuracy rates ranging from 85.4% (aztreonam, small sample size at 41 responses) to 100.0% (three agents). The overall MIC methods accuracy rate was 99.0% among 6,892 responses for the 24 tabulated antimicrobials (Table 2). Few laboratories reported results for three agents that were active against MDR A. baumannii: colistin, polymyxin B, and tigecycline (29 total responses, vast majority generated by the Vitek 2 system). Quantitative methods, such as broth microdilution, are considered the standard for MIC determinations for the polymyxins, since disk diffusion provides unreliable results and zone interpretative criteria are not available. 3 The polymyxin class is an important treatment modality for MDR A. baumannii and representatives of this class should be tested for susceptibility and results reported, as well as those for aminoglycosides, carbapenems, selected other β-lactams, and tetracyclines or their derivatives such as tigecycline.

3 Table 1. Reference test MIC results for ES-01 (2014) sample containing a MDR OXA-23-producing A. baumannii strain originally isolated in Italy. Antimicrobials listed by susceptibility category (Reference MIC in µg/ml): a Susceptible Intermediate Resistant b Amikacin (4) Ceftriaxone (16,32) Amoxicillin / clavulanate (>16) Ceftazidime (4) Minocycline (4,8) Ampicillin (>16) Colistin ( 0.5) Ampicillin / sulbactam (>16) Gentamicin ( 2) Aztreonam (>16) Polymyxin B ( 0.5) Cefazolin (>16) Tigecycline (0.25) Cefepime (>16) TMP / SMX c ( 0.5) Cefoxitin (>16) Tobramycin ( 2) Cefuroxime (>16) Ciprofloxacin (>4) Doripenem (>8) Doxycycline (>8) Ertapenem (>8) Imipenem (>8) Levofloxacin (>4) Meropenem (>8) Moxifloxacin (>4) Nalidixic Acid (>16) Piperacillin (>128) Piperacillin / tazobactam (>64) Tetracycline (>8) Ticarcillin / clavulanate (>128) a. CLSI (2014) interpretive criteria applied [69] where available; for tigecycline the susceptibility breakpoint used was that approved by the USA-FDA for Enterobacteriaceae ( 2 µg/ml). b. Several agents were categorized as resistant due to unmeasurable levels of the MIC, e.g., off-scale values such as >16 µg/ml for amoxicillin / clavulanate. c. TMP / SMX = trimethoprim / sulfamethoxazole.

4 Table 2. Participant performance for selected agents tested by disk agar diffusion (DD) or quantitative MIC methods when applied to the A. baumannii sample ES-01 (2014; only agents with 10 responses for either test method were tabulated). DD MIC Antimicrobial Acceptable category No. % a No. % a Amikacin Susceptible Amoxicillin / clavulanate b - Ampicillin b - Ampicillin / sulbactam Resistant / Intermediate Aztreonam b - Cefazolin b - Cefepime Resistant Cefotaxime Susceptible / Intermediate Cefoxitin b - Ceftazidime c - Ceftriaxone Susceptible / Intermediate Cefuroxime b - Ciprofloxacin Resistant Gentamicin Susceptible Imipenem Resistant Levofloxacin Resistant Meropenem Resistant Nitrofurantoin b, d - Piperacillin Resistant Piperacillin / tazobactam Resistant Tetracycline Resistant Ticarcillin / clavulanate Resistant Tigecycline b, c - Tobramycin Susceptible Trimethoprim b, d - Trimethoprim / sulfamethoxazole Susceptible a. Percentage of categorical responses considered correct by consensus with participant results and reference laboratory values; see Table 1. b. Antimicrobial having no interpretive criteria published in the M100-S24 (2014) document. Categorical report interpretations would be considered inappropriate; see Table 5. c. Consensus categorization could not be achieved (ceftazidime, see Table 3; tigecycline) due to no interpretive breakpoint having been published by CLSI [69] or in the USA-FDA product package insert. d. UTI-specific antimicrobials inappropriately reported for this case scenario of a bacteremia; other UTI drugs reported were norfloxacin (five responses) and sulfisoxazole (one response).

5 Two systems-based testing concerns were detected for cephalosporins (ceftazidime and ceftriaxone) when comparing categorical responses. Table 3 shows the participant responses of five methods / systems for ceftazidime. Three methods indicated a clear activity for ceftazidime (disk diffusion, BD Phoenix, MicroScan; reference MIC at 2-4 µg/ml = susceptible), but the Vitek devices (Vitek 2 and Vitek) demonstrated a lack of drug potency ( % resistant categorization). A review of Vitek responses in 2009 for this strain showed dominant susceptible category findings. Therefore, some modification of the system s interpretive software or breakpoint parameters may have recently occurred leading to these inaccurate results. Table 3. Performance for disk diffusion and four commercial MIC systems for ceftazidime tested against the A. baumannii ES-01 (2014) sample that was susceptible (MIC, 2-4 µg/ml) by reference broth microdilution test results [1]; see Table 1. Method/System (No. responses) % occurrences by category: Susceptible Intermediate Resistant Disk diffusion (22) BD Phoenix (8) MicroScan (340) Vitek (3) a Vitek 2 (234) a a. Results varying from reference MIC values. Similarly, the results for ceftriaxone would have required acceptance of susceptible and intermediate categorical responses (consensus at 80%) to be analyzed for grading, if applied. The ceftriaxone results from Vitek 2 users showed 269 of 288 (93.4%) responses as intermediate, a performance consistent with reference MIC values (Table 1), as well as prior experience with this challenge strain (2009) when using the CLSI 3 breakpoints. In contrast, MicroScan MIC results identified full susceptibility to ceftriaxone for this Acinetobacter spp. strain (249 of 330 responses; 75.5%), clearly an unacceptable categorization (data not shown). Both manufacturers should address these performance aberrations when testing these cephalosporins in their systems against clinical isolates of Acinetobacter spp.

6 Table 4 lists the eleven antimicrobial agents having breakpoint criteria published by both the CLSI 3 and EUCAST. 4 Only one drug (colistin) had identical criteria with most of the other breakpoints generally being one doubling dilution apart. Reporting of results by category requires use of current published breakpoints, and Table 5 lists some reporting issues that may adversely influence patient treatment / outcomes. Local standard operating procedures should be instituted to prevent these serious reporting errors. Table 4. Comparative breakpoint criteria when testing Acinetobacter species (CLSI and EUCAST, 2014) a. Antimicrobial agent Susceptible/Resistant (µg/ml) MIC criteria for: CLSI (2014) EUCAST (2014) Doripenem 2 / 8 1 / 4 Imipenem 2 / 8 2 / 16 Meropenem 2 / 8 2 / 16 Colistin 2 / 4 b 2 / 4 b Amikacin 16 / 64 8 / 32 Gentamicin 4 / 16 4 / 8 Netilmicin 8 / 32 4 / 8 Tobramycin 4 / 16 4 / 8 Ciprofloxacin 1 / 4 1 / 2 Levofloxacin 2 / 8 1 / 4 Trimethoprim / Sulfamethoxazole 2 / 4 2 / 8 a. Most recent published breakpoint criteria [3,4]. b. Eleven drugs have breakpoints to compare between organizations, and only one (colistin) had identical criteria for susceptible and resistant (9.1%).

7 Table 5. List of testing and reporting concerns observed for this Acinetobacter bacteremia case scenario. 1) Antimicrobials tested / reported that were not appropriate for a bloodstream infection (59 responses) Nitrofurantoin, norfloxacin, sulfonamides and trimethoprim (UTI only agents) 2) Reporting of Gram-positive focused agents (13 responses) Azithromycin, clindamycin, erythromycin, linezolid, oxacillin, penicillin, rifampin, and vancomycin (no spectrum of activity versus Acinetobacter spp.) 3) Testing and reporting categorized results where no CLSI (2014) interpretive criteria exist (278 responses) Amoxicillin/clavulanate, ampicillin, aztreonam, cefaclor, cefazolin, cefdinir, cefixime, cefoperazone, cefoxitin, cefpodoxime, ceftizoxime, cefuroxime, cephalothin, chloramphenicol, ertapenem, gemifloxacin, moxifloxacin, ofloxacin and tigecycline Results were reported from disk diffusion and MIC methods/systems (MicroScan, BD Phoenix, Vitek 2, Vitek products). Finally, a comparison of participant methods used in 2014 versus 2009 illustrates 6.6% more responding laboratories, the vast majority (96.2%) of which utilized MIC-type commercial systems. This is an increase in MIC use from 94.5% in 2009, and only 3.8% of participants now use the DD method. Emerging Resistance and Epidemiology Among Acinetobacter spp. Recent surveillance data shows that approximately 60% of healthcare-associated A. baumannii in the United States are MDR. This organism is able to survive on human skin and dry surfaces, possesses an array of chromosomal or plasmid-borne resistance genes, and can also readily acquire foreign DNA. 10 These characteristics allow Acinetobacter spp. the ability to efficiently respond to antimicrobial selective pressure. 11,12 As an example, analysis of the genome of an epidemic A. baumannii strain from France (AYE) demonstrated an 86-kb island, where 86.5% was comprised by antimicrobial resistance determinants. Moreover, the vast majority of the open reading frames have originated from other Gram negative organisms and the G + C content of this region was higher (52.8%) than the remaining chromosome (38.8%), evidence of DNA acquisition. 8 Another recent study analyzing the genomes of A. baumannii isolates from a university hospital in Cleveland, Ohio indicated the existence of a local endemic and interacting population of A. baumannii either within the hospital system or in the colonized patients. 10

8 Antimicrobial resistance in A. baumannii can be due to enzymatic hydrolysis, modified penicillin-binding proteins, and decreased permeability by efflux-pump systems or altered porin proteins The inherent chromosomally encoded AmpC cephalosporinase in A. baumannii is the most common resistance mechanism directed against β-lactams. 15 This resistance determinant, also known as Acinetobacterderived cephalosporinase (ADC), belongs to the Class C β-lactamases and hydrolyzes penicillins and narrow- and extended-spectrum cephalosporins, but not cefepime or carbapenems. 12 Although transcription of ADC-encoding genes is not inducible as observed in other Gram negative organisms, the presence of insertion sequence (IS) elements, such as ISAba1, located upstream of these genes has been found to provide promoter regions and increased AmpC expression. 16 Other β-lactamases such as TEM- and SHV-type, PER, and VEB have been reported in A. baumannii. Among Class A enzymes detected in A. baumannii, PER enzymes seem to be the most common and very prevalent in isolates from Korea and Turkey, and have also been observed in organisms recovered from several other countries such as France, Belgium, Romania, Hungary, Bulgaria, Russia, India, China, and United States The above listed Class A enzymes do not confer a carbapenem resistance phenotype, and among those with carbapenemase activity (KPC, GES, SME, NMC and IMI), KPC and 14, 25, 26 GES have yet been reported in A. baumannii. A. baumannii produce a second intrinsic group of β-lactamases represented by Class D OXA-51/69-like enzymes, 27 which possess a limited hydrolytic activity towards carbapenems, but the insertion of ISAba1 28, 29 in the upstream region may contribute to a resistance phenotype to these compounds. Moreover, a report of a new ISAba9 detected upstream of ISAba1/bla OXA-51 in isolates from Greece provided additional promoter regions and seemed to further enhance carbapenem resistance. 30 These IS not only provide increased transcription levels for downstream genes, but also possess the ability to mobilize these genes in in vitro experiments. 31 This species-specific group of OXA-51/69-like-encoding genes has recently been detected in a plasmid of an Acinetobacter genome species 13TU A. nosocomialis, evidence of in vivo mobilization. 32 Most worrisome has been the worldwide dissemination of acquired carbapenem-hydrolyzing Class D β-lactamases among clinical isolates of A. baumannii, which has been described in numerous reports These enzymes are represented by three distinct groups (OXA-23, OXA-24/40 and OXA-58, OXA-143), and although they possess generally low hydrolytic activity towards carbapenems, it has been 42, 43 demonstrated that these enzymes significantly contribute to carbapenem resistance. This contribution is mainly, as with OXA-51/69-like enzymes, due to the presence of IS elements providing promoter regions (ISAba1 upstream of bla OXA-23 and ISAba1, 2, or 3 upstream of bla OXA-58 ) and consequently, elevated expression levels. 25

9 Four groups of acquired Class B metallo-β-lactamase (MBL)-encoding genes (bla IMP - and bla VIM -like, bla SIM, and more recently bla NDM ) have been reported in Acinetobacter spp. 44 bla IMP variants have been detected among A. baumannii in Brazil, Italy, Portugal, Japan, South Korea, China, Taiwan, and Australia, 20, 25, , 25 while bla VIM -like and bla SIM-1 have been more limited to Asian-Pacific countries. The more recent bla NDM-1 and bla NDM-2 allelic variants have been found among Acinetobacter spp. in numerous countries worldwide. Despite being less prevalent than acquired carbapenem-hydrolyzing Class D 8, 13 β-lactamases, MBL variations possess significantly more carbapenem hydrolytic activity. In addition, MBL genes are observed in the variable region of genetic structures (integrons), usually associated with 8, 13, 45 other resistant determinants such as aminoglycoside-modifying enzymes. Compared to other Gram negative organisms, not much is known about resistance mechanisms in A. baumannii due to decreased antimicrobial permeability (reduction of transport into the periplasmic space). Previous studies report that carbapenem resistant isolates of A. baumannii had reduced expression of 47, 44, and 37 kda porin proteins. 48 More recently, the loss of a 29-kDa protein (CarO) 8, 48 has been associated with imipenem and meropenem resistance. Regarding efflux-pump systems in A. baumannii, AdeABC, AdeIJK, and AdeFGH pumps have been demonstrated to provide a MDR phenotype by actively expelling aminoglycosides, cefotaxime, tetracyclines, erythromycin, 12, 14 chloramphenicol, trimethoprim, and fluoroquinolones. AdeABC represents the most frequent system (53 to 97%) associated with MDR in clinical strains, and it has not been detected in environmental isolates. 14 Treatment of Acinetobacter Infections Acinetobacter spp. are typically opportunistic pathogens and it may be difficult to determine if their 5, 8, 49 presence indicates colonization or infection. As Acinetobacter spp. have the ability to survive in a wide range of environments as well as surviving on surfaces for long periods of time, they may be the cause of outbreaks and become endemic in the health-care setting. 50 In the hospital, Acinetobacter spp. are found in bloodstream infections, ventilator-associated pneumonia (VAP), urinary tract infections, and skin and soft tissue infections. 50 A. baumannii bloodstream infections and VAP have been associated 5, with a high level of mortality. A. baumannii are frequently MDR and there are a relatively limited number of antimicrobial agents which 12, 50, 51 retain activity against such strains. In fact, there are only five antimicrobial classes recommended for testing against Acinetobacter spp. by both EUCAST and CLSI. Those agents are the carbapenems (doripenem, imipenem, and meropenem), the lipopeptides or polymyxins (colistin), the aminoglycosides (amikacin, gentamicin, netilmicin, and tobramycin), the fluoroquinolones (ciprofloxacin and levofloxacin), and the folate antagonists (trimethoprim-sulfamethoxazole). Surprisingly, colistin is the only agent of the

10 eleven listed (Table 4) to have the identical interpretive criteria in the EUCAST and CLSI documents. A. baumannii are generally resistant to penicillin, ampicillin, first-generation cephalosporins, gentamicin, and chloramphenicol. 54 Sulbactam (a β-lactamase inhibitor) has intrinsic activity and has shown some treatment efficacy The emergence of increasing resistance in A. baumannii to aminoglycosides and carbapenems has led to the presence of strains for which colistin (or polymyxin B) and tigecycline may be 12, 51, 54, 58 needed. As there is concern that resistance to tigecycline may develop by upregulation of efflux pumps and an inability to reach adequate peak serum concentrations, tigecycline may be more focused 8, for salvage therapy. Carbapenems are recommended for susceptible strains ; for MDR strains, 50, 62 colistin plus imipenem or meropenem. Alternative therapy may be ampicillin-sulbactam or intravenous minocycline used alone or in various combinations. 62 Due to the extensive MDR problem, a number of studies both in vitro and in vivo have examined a variety of antimicrobial combinations with varying degrees of success. 50 Included among the combination therapies have been combinations of colistin and rifampin; meropenem and rifampin; imipenem and rifampin; imipenem and amikacin; and colistin plus meropenem or imipenem, or an aminoglycoside, or 50, 58, ampicillin-sulbactam, or ciprofloxacin. Although there are currently a number of in vitro and some uncontrolled clinical case studies, there exists a lack of well-controlled comparative trials with candidate 50, 68 combinations to provide definitive treatment recommendations. References 1. Clinical and Laboratory Standards Institute M07-A9. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard: ninth edition. Clinical and Laboratory Standards Institute, Wayne, PA. 2. Clinical and Laboratory Standards Institute M02-A11. Performance standards for antimicrobial disk susceptibility tests; approved standard: eleventh edition. Clinical and Laboratory Standards Institute, Wayne, PA. 3. Clinical and Laboratory Standards Institute M100-S24. Performance standards for antimicrobial susceptibility testing: 24th informational supplement. Clinical and Laboratory Standards Institute, Wayne, PA. 4. European Committee on Antimicrobial Susceptibility Testing Breakpoint tables for interpretation of MICs and zone diameters. Version 4.0, January Available at Accessed January 1, Versalovic J, Carroll KC, Funke G, Jorgensen J, Landry ML, Warnock DW Manual of Clinical Microbiology, 10th ed. ASM Press, Washington D.C.

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