ANTIMICROBIAL SUSCEPTIBILITY – TESTING NEWLY RECOGNIZED 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 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 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

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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 1 100.0 288 92.4 Cefoxitin Susceptible 2 100.0 314 93.6 Ceftazidime Susceptible 9 100.0 542 97.8 Ceftriaxone Susceptible 19 100.0 726 96.7 Cefuroxime Susceptible - Intermediate 4 100.0 352 86.1 Ciprofloxacin Susceptible 25 96.0 747 99.3 Ertapenem Susceptible 0 -- 523 99.6 Gentamicin Susceptible 14 100.0 839 99.3 Imipenem Susceptible 5 100.0 493 99.2 Levofloxacin Susceptible 8 100.0 761 99.5 Meropenem Susceptible 1 100.0 377 99.5 Nitrofurantoin Susceptible 23 100.0 761 93.8 Piperacillin-Tazobactam Susceptible 7 100.0 725 99.7 Tetracycline Susceptible - Intermediate 11 100.0 353 98.0 Ticarcillin-Clavulanate Susceptible 2 100.0 100 98.0 Tobramycin Susceptible 7 100.0 657 99.4 TMP-SMXb Susceptible 27 100.0 780 99.5 a. Correct categorical interpretation was determined by the reference MIC using the M07-A10 method,1 and CLSI M100-S27, EUCAST and USCAST breakpoint criteria,3-5 where available (exception tigecycline).6 b. TMP-SMX = trimethoprim-sulfamethoxazole

and nitrofurantoin. The numbers of responses in this subset analysis by method/product were: DD (3-16 responses/drug; average 10), MicroScan (174-192 responses/drug; average 180) and Vitek 2 (168-203 response/drug; average 186). The expected rate of susceptible category responses was uniformly lower for the MicroScan devices (-0.6 to -12.0%; average = -3.3%) compared to Vitek 2. Vitek 2 and DD susceptible rates were not significantly different, but analysis was limited due to the smaller number of DD responses. False resistance appears to be more likely for MicroScan products across these tested antimicrobial classes.

Beta-Lactamases and Other Resistance Mechanisms in Klebsiella spp.

In general, K. variicola isolates are susceptible to most clinically used antimicrobial agents. In a study evaluating 420 clinical isolates from 26 hospitals originally identified as K. pneumoniae, it was observed

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ANTIMICROBIAL SUSCEPTIBILITY – TESTING NEWLY RECOGNIZED KLEBSIELLA SPECIES AND ASSOCIATED ANTIBIOGRAMS (cont.)

that phylogenetic groups of KpI (K. pneumoniae sensu stricto), KpII (K. quasipneumoniae), and KpIII (K. variicola) were widespread geographically, although K. pneumoniae sensu stricto was the most common organism comprising 80.3% of the isolates.10 The same study compared the susceptibility patterns among the K. pneumoniae phylogenetic groups when tested against several antimicrobial agents. For all agents, the level of resistance was highest in K. pneumoniae sensu stricto, intermediate in K. quasipneumoniae, and lowest in K. variicola.10 K. variicola resistance levels were lowest for ceftriaxone, ceftazidime, piperacillin-tazobactam, gentamicin, amikacin and ciprofloxacin when compared to other phylogenetic groups tested; also see Table 3.

More recently, K. variicola isolates carrying carbapenemase-encoding genes have been described, including genes encoding OXA-181,14 NDM-915 and IMI-2;16 however, only the IMI-2-producing isolate was detected in a clinical specimen. In the other instances, K. variicola was detected in environmental or food samples; nevertheless, these reports highlight for the possibility of this organism acquiring resistance genes and becoming a challenge for antimicrobial treatment.

Other common β-lactamase genes (blaSHV, blaKPC and blaCTX-M) have been observed among K. pneumoniae, K. oxytoca and other related species such as Raoultella spp.17-19 These genes have not been documented in K. variicola, but exchange of these mobile genes between related species remains common and would produce resistance to penicillins and cephalosporins as well as the carbapenems in

the case of blaKPC.

K. variicola isolates are usually susceptible to polymyxins, but this isolate (ES-02 2017) was resistant to colistin (MIC, 8 µg/ml). Klebsiella species usually become resistant to the polymyxins following alterations in the lipopolysaccharide that constitutes the bacterial cell wall.20 Mutations or deleterious insertions in the genes that modify the lipopolysaccharide, such as mgrB, phoP, phoQ, pmrA and pmrB, encode polymyxin resistance in clinical isolates of Klebsiella spp.20 Disruptions on mgrB were recently shown to be a common colistin resistance mechanism in K. pneumoniae clinical isolates.21

The recent discovery of a plasmid-mediated gene encoding polymyxins resistance has been regarded as a major threat to public health.22 These genes, named mcr (mobile colistin resistance gene), encode low- level colistin resistance and were initially reported in Escherichia coli; but K. pneumoniae isolates carrying mcr-like genes have been described in several countries. As with other mobile resistance genes, mcr-like could be transferred to a K. variicola isolate, although ES-02 (2017) was negative for the presence of these genes.

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ANTIMICROBIAL SUSCEPTIBILITY – TESTING NEWLY RECOGNIZED KLEBSIELLA SPECIES AND ASSOCIATED ANTIBIOGRAMS (cont.)

Table 3. MIC distributions for 13 antimicrobial agents tested against 73 K. variicola strains isolates USA hospitals (SENTRY Antimicrobial Surveillance Program, 2015-2016).a

Cum. % of tested isolates inhibited at MIC (µg/ml): MIC % Antimicrobial agent ≤0.06 0.12 0.25 0.5 1 2 4 8 16 50% 90% Susceptibleb Amikacin -- -- 1.4 8.2 69.9 94.5 97.3 97.3a 100.0a 1 2 100.0 / 97.3 Aztreonem -- 79.5 87.7 90.4 90.4 90.4 90.4 90.4 94.5 ≤0.12 0.5 90.4 / 90.4 Cefepime -- 84.9 90.4 91.8 93.2 93.2 93.2 93.2 93.2 ≤0.12 0.25 93.2 / 93.2 Ceftazidime 13.7 52.1 75.3 83.6 89.0 90.4 93.2 93.2 93.2 0.12 2 93.2 / 89.0 Ceftriaxone 65.8 78.1 86.3 89.0 89.0 90.4 90.4 90.4 -- ≤0.06 2 89.0 / 89.0 Colistin 1.4 56.2 79.5 82.2 91.8 93.2 93.2 93.2 -- 0.12 1 93.2 / 93.2 Gentamicin 1.4 5.5 58.9 91.8 93.2 93.2 93.2 97.3 -- 0.25 0.5 93.2 / 93.2 Levofloxacin 64.4 75.3 78.1 91.8 95.9 95.9 98.6 -- -- 0.06 0.5 95.9 / 91.8 Meropenem 97.3 97.3 98.6 98.6 98.6 98.6 98.6 98.6 98.6 0.03 0.03 98.6 / 98.6 Piperacillin-Tazobactam ------4.1 13.7 54.8 78.1 83.6 90.4 2 16 90.4 / 83.6 Tetracycline -- -- 1.4 6.8 67.1 86.3 90.4 94.5 97.3 1 4 90.4 / -- Tobramycin 1.4 5.5 56.2 93.2 93.2 93.2 93.2 93.2 -- 0.25 0.5 93.2 / 93.2 TMP-SMX ------91.8 91.8 94.5 94.5 -- -- ≤0.5 ≤0.5 94.5 / 94.5 a. SENTRY Program results kindly provided by JMI Laboratories (North Liberty, Iowa USA) b. % susceptibility by CLSI/EUCAST breakpoints,3,4 see underlined values. Percentage susceptibility can vary between breakpoint organization criteria from nil (9 drugs) to 6.8% (piperacillin-tazobactam).

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ANTIMICROBIAL SUSCEPTIBILITY – TESTING NEWLY RECOGNIZED KLEBSIELLA SPECIES AND ASSOCIATED ANTIBIOGRAMS (cont.)

Treatment for Klebsiella spp. in UTIs

UTIs are one of the most frequent infections encountered in both inpatients and outpatient settings, and represents a major source of Gram-negative bacteremia. Both Enterobacteriaceae and non-fermentative Gram-negative bacilli are important causes of UTI, but E. coli is by a large margin the most frequent pathogen causing community as well as health-care associated UTIs. Other Enterobacteriaceae species, such as K. pneumoniae, K. oxytoca, Proteus mirabilis Enterobacter cloacae and Serratia marcescens, also represent important causes of UTI. In recurrent UTI, especially in the presence of urinary tract structural abnormalities, the relative frequency increases for Klebsiella spp., Proteus spp., Enterobacter spp., Pseudomonas aeruginosa and Acinetobacter spp.23

Treatment of UTI has been the subject of many studies and guidelines as rates of antimicrobial resistance have evolved.17,24,25 When dealing with recurrent and/or complicated UTI, common measures include obtaining a urine culture, starting broad-spectrum antimicrobial coverage and then refining the drug selection after receiving susceptibility testing results.25 The recent emergence of ESBL producers among community-acquired Klebsiella spp. and other Enterobacteriaceae UTIs is troubling due to the limited orally delivered treatment options available for these organisms.24 Furthermore, after instrumentation and/or repeat courses of antimicrobial therapy, antimicrobial-resistant isolates might be expected.25

The major challenge for clinicians is to combine local susceptibility patterns with the agents that are most likely to be effective. Targeted and appropriate antimicrobial therapy can significantly reduce the morbidity and mortality associated with this infection type. However, antimicrobial resistance patterns can vary substantially by geographic region or even between institutions within a region. Recommendations form the International Clinical Practice Guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women includes nitrofurantoin (100 mg twice daily for 5 days), fosfomycin trometamol (3 grams in a single dose), TMP-SMX (160/800 mg twice daily for 3 days), fluoroquinolones in 3 day regimens, and β-lactam agents (amoxicillin-clavulanate, cefdinir, and cefpodoxime-proxetil) in 3-7 day regimens.25 However, TMP-SMX, currently available fluoroquinolones and the β-lactam agents listed (above) in the guidelines should not be recommended for empiric therapy of recurrent UTI in many geographic regions due to increased resistance.24,25 In contrast, nitrofurantoin and fosfomycin appear to remain as reliable orally administered options for empirical UTI treatment.17

Other antimicrobial agents more recently approved for treatment of complicated UTI include ceftolozane- tazobactam and ceftazidime-avibactam.26-30 These two β-lactamase inhibitor combinations represent valuable additions to the armamentarium for treatment of serious UTIs, especially those caused by multidrug-resistant (MDR) Gram-negative bacilli. All of the above agents would represent appropriate choices for treatment of the K. variicola strain isolated in this simulated case (ES-02 2017), as it was generally susceptible to all agents with the notable exceptions of ampicillin and colistin. Data found in

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ANTIMICROBIAL SUSCEPTIBILITY – TESTING NEWLY RECOGNIZED KLEBSIELLA SPECIES AND ASSOCIATED ANTIBIOGRAMS (cont.)

Table 3 documents the following antimicrobial susceptibility rates3,4 for recently identified K. variicola isolates when tested against the more potent agents: aminoglycosides (93.2-100.0%), colistin (91.8%), fluoroquinolones (91.8-95.9%), piperacillin-tazobactam (83.6-90.4%) and TMP-SMX (94.5%). The antimicrobial coverage of these 73 K. variicola by the newest beta-lactamase inhibitor combinations ranged from 94.5% (ceftolozane-tazobactam) to 100.0% (ceftazidime-avibactam; data not shown).26,27

References

1. CLSI. M07-A10. Methods for dilution antimicrobial susceptibility tests for that grow aerobically; approved standard- tenth edition. Wayne, PA: Clinical and Laboratory Standards Institute 2015.

2. CLSI. M02-A12. Performance standards for antimicrobial disk susceptibility tests; Twelfth Edition. Wayne, PA: Clinical and Laboratory Standards Institute 2015.

3. CLSI. M100-S27. Performance standards for antimicrobial susceptibility testing: 27th informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute 2017.

4. EUCAST (2017). Breakpoint tables for interpretation of MICs and zone diameters. Version 7.0, January 2017. Available at http://www.eucast.org/clinical_breakpoints/. January 2017.

5. USCAST (2017). Breakpoint tables for interpretations of MICs and Zone Diameters, Version 3.0, January 2017. Available at http://www.uscast.org/breakpoints.html. March 2017.

6. Tygacil. Tygacil ® Package Insert. Package Insert. 2016 Accessed at www.tygacil.com on February 23, 2017.

7. Jorgensen JH, Pfaller MA, Carroll KC, et al. Manual of Clinical Microbiology, 11th ed. Washington, D.C.: ASM Press, 2015.

8. Garza-Ramos U, Silva-Sanchez J, Martinez-Romero E, et al. Development of a multiplex-PCR probe system for the proper identification of Klebsiella variicola. BMC Microbiol. 2015; 15:64.

9. Rosenblueth M, Martinez L, Silva J, Martinez-Romero E. Klebsiella variicola, a novel species with clinical and plant-associated isolates. Syst Appl Microbiol. 2004; 27:27-35.

10. Brisse S, Passet V, Grimont PA. Description of Klebsiella quasipneumoniae sp. nov., isolated from human infections, with two subspecies, Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and Klebsiella quasipneumoniae subsp. similipneumoniae subsp. nov., and demonstration that Klebsiella singaporensis is a junior heterotypic synonym of Klebsiella variicola. Int J Syst Evol Microbiol. 2014; 64:3146-3152.

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11. Berry GJ, Loeffelholz MJ, Williams-Bouyer N. An investigation into laboratory misidentification of a bloodstream Klebsiella variicola infection. J Clin Microbiol. 2015; 53:2793-2794.

12. Fonseca EL, Ramos ND, Andrade BG, Morais LL, Marin MF, Vicente AC. A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae in the clinical routine. Diagn Microbiol Infect Dis. 2017; 87:315-317.

13. Maatallah M, Vading M, Kabir MH, et al. Klebsiella variicola is a frequent cause of bloodstream infection in the stockholm area, and associated with higher mortality compared to K. pneumoniae. PLoS One. 2014; 9:e113539.

14. Zurfluh K, Poirel L, Nordmann P, Klumpp J, Stephan R. First detection of Klebsiella variicola producing OXA-181 carbapenemase in fresh vegetable imported from Asia to Switzerland. Antimicrob Resist Infect Cont. 2015; 4:38.

15. Di DY, Jang J, Unno T, Hur HG. Emergence of Klebsiella variicola positive for NDM-9, a variant of New Delhi metallo-beta-lactamase, in an urban river in South Korea. J Antimicrob Chemother. 2017; 72:1063-1067.

16. Hopkins KL, Findlay J, Doumith M, et al. IMI-2 carbapenemase in a clinical Klebsiella variicola isolated in the UK. J Antimicrob Chemother. 2017:in press.

17. Hisano M, Bruschini H, Nicodemo AC, Gomes CM, Lucon M, Srougi M. The bacterial spectrum and antimicrobial susceptibility in female recurrent urinary tract infection: How different they are from sporadic single episodes? Urology. 2015; 86:492-497.

18. Sigler M, Leal JE, Bliven K, Cogdill B, Thompson A. Assessment of appropriate antibiotic prescribing for urinary tract infections in an internal medicine clinic. Southern medical journal. 2015; 108:300-304.

19. Sastry S, Clarke LG, Alrowais H, Querry AM, Shutt KA, Doi Y. Clinical appraisal of fosfomycin in the era of antimicrobial resistance. Antimicrob Agents Chemother. 2015; 59:7355-7361.

20. Olaitan AO, Morand S, Rolain JM. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front Microbiol. 2014; 5:643.

21. Cannatelli A, Giani T, D'Andrea MM, et al. MgrB inactivation is a common mechanism of colistin resistance in KPC-producing Klebsiella pneumoniae of clinical origin. Antimicrob Agents Chemother. 2014; 58:5696-5703.

22. Liu YY, Wang Y, Walsh TR, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016; 16:161-168.

23. Lane DR, Takhar SS. Diagnosis and management of urinary tract infection and pyelonephritis. Emergency medicine clinics of North America. 2011; 29:539-552.

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24. Briongos-Figuero LS, Gomez-Traveso T, Bachiller-Luque P, et al. Epidemiology, risk factors and comorbidity for urinary tract infections caused by extended-spectrum beta-lactamase (ESBL)- producing enterobacteria. Int J Clin Pract. 2012; 66:891-896.

25. Gupta K, Hooton TM, Naber KG, et al. International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: A 2010 update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases. Clin Infect Dis. 2011; 52:e103-e120.

26. AVYCAZ®. AVYCAZ® (ceftazidime-avibactam). Irvine, CA: Allergan USA, Inc. 2017. Available at https://www.allergan.com/assets/pdf/avycaz_pi. Date Accessed February 2017.

27. ZERBAXA. ZERBAXA® (ceftolozane/tazobactam). package insert. 2016 Accessed at https://www.merck.com/product/usa/pi_circulars/z/zerbaxa/zerbaxa_pi.pdf on January 2017.

28. Zhanel GG, Lawson CD, Adam H, et al. Ceftazidime-avibactam: a novel cephalosporin/β-lactamase inhibitor combination. Drugs. 2013; 73:159-177.

29. Bush K. A resurgence of beta-lactamase inhibitor combinations effective against multidrug-resistant Gram-negative pathogens. Int J Antimicrob Agents. 2015; 46:483-493.

30. Sader HS, Farrell DJ, Castanheira M, Flamm RK, Jones RN. Antimicrobial activity of ceftolozane/tazobactam tested against Pseudomonas aeruginosa and Enterobacteriaceae with various resistance patterns isolated in European hospitals (2011-2012). J Antimicrob Chemother. 2014; 69:2713-2722.

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