ANTIMICROBIAL SUSCEPTIBILITY – TESTING NEWLY RECOGNIZED KLEBSIELLA SPECIES AND ASSOCIATED ANTIBIOGRAMS Sample ES-02 (2017) was a simulated urinary tract culture for organism identification and susceptibility testing using laboratories' routinely applied methods.1-6 The patient was a hospitalized 38-year-old female having symptoms of a urinary tract infection (UTI). The sample contained a Klebsiella variicola organism in pure culture having a generally wildtype antibiogram, but with an unusual resistance to polymyxin class agents (colistin, polymyxin B). Furthermore, modest elevations in tetracycline class agents were observed; however, no evidence of an extended-spectrum β-lactamase (ESBL) was discovered. This sample was distributed as an ungraded educational challenge to determine the ability of currently used susceptibility testing products to recognize and appropriately categorize antimicrobial activity among less commonly recognized Enterobacteriaceae species causing UTIs. Organism Identification Organism identification responses of K. variicola (none; 0.0%), K. pneumoniae (895; 96.5%), Klebsiella spp. (eight; 0.9%) and Gram-negative organism (eight; 0.9%) were considered acceptable identification performance (98.3% overall; see below). The most common erroneously reported species-level identifications were K. oxytoca (seven) and various Pseudomonas spp. (three). Correct, to species identifications (K. variicola) was currently near zero among commercial devices. K. pneumoniae results were noted for the following systems/devices (% observed for > 10 responses): MicroScan (97.2%), Vitek 2 (98.1%) and manual methods (81.1%). This level of contemporary accuracy was considered very compromised overall, and only one reference laboratory site now using the MALDI-TOF device, had the correct K. variicola response. The genus Klebsiella is comprised of encapsulated Gram-negative non-motile rods within the family Enterobacteriaceae, which reside in the upper respiratory tract and the gastrointestinal tract of mammals. The most common clinically isolated species are Klebsiella pneumoniae followed by Klebsiella oxytoca.7 This pathogen group has been reported to represent 7-10% of all hospital-acquired infections in North America, Europe and Latin America.8 In 2004, a new species, Klebsiella variicola, was described based on the phylogenetic analysis of rpoB, gyrA, mdh, infB, phoE and nifH genes and DNA-DNA hybridizations.9 K. variicola has been isolated from clinical and environmental sources, with clinical strains most often originating from bloodstream and urinary tract infections.7 The work presented by Rosenblueth et al. demonstrated that K. variicola represented less than 10% of clinical isolates previously identified as K. pneumoniae.9 Since that report, isolates historically identified as K. pneumoniae have been subdivided into phylogenetic groups named KpI (K. pneumoniae sensu stricto), KpII (K. quasipneumoniae), and KpIII (K. variicola), which usually cannot be differentiated with current commercial systems.10 One phenotypic difference between K. variicola and K. pneumoniae is that greater than 95% of K. variicola isolates do not ferment the carbohydrate adonitol, whereas most K. pneumoniae isolates will ferment adonitol.11 Unfortunately, it is ANTIMICROBIAL SUSCEPTIBILITY – TESTING NEWLY RECOGNIZED KLEBSIELLA SPECIES AND ASSOCIATED ANTIBIOGRAMS (cont.) unable to differentiate K. variicola from K. quasipneumoniae, which are both typically adonitol negative.12 Klebsiella variicola and K. quasipneimoniae are not included in the databases of most commercial identification systems (Vitek 2, MicroScan) and share similar biochemical and mass spectrometry profiles, which leads to misidentification within the clinical microbiology laboratory.8 Thus, the clinical importance of K. variicola and K. quasipneumoniae may be underestimated. A recent study indicated K. variicola was associated with a higher mortality rate in bloodstream infections when compared to K. pneumoniae.13 Therefore, accurate identification would be essential to fully investigate the clinical significance of this emerging species. The most reliable way to differentiate K. variicola from other Klebsiella species is by genotypic methods, such as rpoB sequencing.7,10 Recently, a one-step multiplex PCR method was reported to discriminate K. pneumoniae, K. variicola and K. quasipneumoniae, which would enhance more rapid results in a clinical setting compared to traditional sequencing methods.12 Antimicrobial Susceptibility Testing (Ungraded) Participants were asked to perform antimicrobial susceptibility testing on this K. variicola isolate. This strain was selected to challenge proper identification and to determine antimicrobial coverage across numerous classes of antimicrobial agents that are active against Enterobacteriaceae. The initial reference laboratory antimicrobial susceptibility testing was conducted using the standardized reference broth microdilution method,1 and susceptibility categories were determined by applying CLSI and other recognized international criteria,3-6 where available. The reference laboratory testing reported a total of 20 agents (Table 1) that demonstrated significant activity (susceptible categorical result) against this strain; however, the K. variicola was less susceptible to penicillins (ampicillin), a common finding among Klebsiella species. The colistin resistance was unusual, and no susceptibility test results for this antimicrobial were reported by participating laboratories. Consensus participant MIC categorical accuracy ranged from 86.1% (cefuroxime) to 99.7% (piperacillin- tazobactam) with three agents (ampicillin-sulbactam, cefuroxime, tetracyclines) having reference MIC or consensus categorical values at or near the "intermediate plus susceptible" concentrations (Tables 1 and 2). The disk diffusion (DD) results, though much smaller in number (≤27 for each drug), had an overall categorical accuracy ranging from 91.7 (cefazolin) to 100.0% (19 drugs; Table 2). Table 2 results demonstrate the generally susceptible antibiogram for this K. variicola isolate observed by the participants. The intrinsic, usually enzyme-mediated, resistance to aminopenicillin (ampicillin) was confirmed by 95.7% (DD) to 97.9% (MICs) of reported values. Cefuroxime and the tetracyclines (doxycycline, minocycline, tetracycline HCL) were less potent e.g., susceptible or intermediate category. Some agents with high-level activity were rarely tested and reported: ceftazidime-avibactam (three American Proficiency Institute - 2017 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY – TESTING NEWLY RECOGNIZED KLEBSIELLA SPECIES AND ASSOCIATED ANTIBIOGRAMS (cont.) Table 1. Listing of expected reference susceptibility testing categorical results for this K. variicola sent as ungraded sample ES-02 (2017). Antimicrobials listed by susceptibility category (Reference MIC in µg/ml)a Susceptible Intermediate Resistant Amikacin (2) Ampicillin-Sulbactam (16/8) Ampicillin (>16) Aztreonam (0.5) Tetracycline (8) Colistin (8) Cefepime (0.25) Ceftaroline (0.25) Ceftazidime (1) Ceftazidime-Avibactam (0.5/4) Ceftolozane-Tazobactam (1/4) Ceftriaxone (0.12) Ciprofloxacin (0.12) Doripenem (0.12) Doxycycline (4) Gentamicin (0.5) Imipenem (0.5) Levofloxacin (0.12) Meropenem (0.03) Minocycline (4) Piperacillin-Tazobactam (8/4) Tigecycline (1) Tobramycin (0.5) TMP-SMX (≤0.5/9.5) a. Susceptibility categories determined by CLSI M100-S27 (2017)3-5 b. TMP-SMX = trimethoprim-sulfamethoxazole MicroScan responses), ceftolozane-tazobactam (none) and fosfomycin (none). Yet other participants reported antimicrobial agents (azithromycin, clarithromycin, linezolid, moxifloxacin, penicillin, telithromycin, vancomycin) inappropriate for this sample or infection site. One to 356 categorical responses were reported for oral cephalosporins (cefaclor, cefixime, cefpodoxime, cefuroxime). Each of these compounds could have had their susceptible category result predicted by the cefazolin surrogate test (789 responses; 91.7-96.3% susceptible) at a susceptible MIC breakpoint of ≤16 µg/ml.3,5 Cross resistance is not predicted by this surrogate test and participants should refer to the breakpoint standard publication used by your laboratory for a complete list of orally delivered cephalosporin agents predicted by cefazolin inhibition results.3-5 Finally, the susceptibility rates of eight agents representing seven antimicrobial classes were analyzed to detect potential differences by method used (DD, MicroScan, Vitek 2). The drug classes (tested agent) were: penicillins (ampicillin), cephalosporins (cefazolin, ceftriaxone), fluoroquinolones (ciprofloxacin), aminoglycosides (gentamicin), β-lactamase inhibitor combinations (piperacillin-tazobactam), TMP-SMX American Proficiency Institute - 2017 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY – TESTING NEWLY RECOGNIZED KLEBSIELLA SPECIES AND ASSOCIATED ANTIBIOGRAMS (cont.) Table 2. Participant performance for 24 selected agents (>100 response by one or both tests) listed by agar disk diffusion (DD) and quantitative MIC methods for ES-02 (2017), a K. variicola UTI isolate. DD MIC Antimicrobial agent Acceptable categorya No. % correct No. % correct Amikacin Susceptible 4 100.0 398 99.5 Amoxicillin-Clavulanate Susceptible 15 93.3 420 99.3 Ampicillin Resistant 23 95.7 778 97.9 Ampicillin-Sulbactam Susceptible - Intermediate 2 100.0 695 96.5 Aztreonam Susceptible 2 100.0 341 95.3 Cefazolin Susceptible 12 91.7 777 96.3 Cefepime Susceptible 2 100.0 612 96.6 Cefotaxime Susceptible