ANTIMICROBIAL SUSCEPTIBILITY INCREASING COMPLEXITY FOR TREATMENT OF URINARY

Transcription

1 TRACT INFECTIONS This specimen was stated to be "from a urine culture" and was to be identified to the species level and tested for antimicrobial susceptibility. The culture contained an Escherichia coli commonly associated with urinary tract infections in the ambulatory (outpatient) and hospital settings. The strain was selected to highlight several emerging resistance mechanisms (discussed below) and was distributed by API to survey participants as an Educational Sample (ES) challenge and susceptibility testing sample. Participant grading was not performed. Responses of E. coli and E. coli, ESBL strain combined for a percentage of 98.0% of responses. Other acceptable identifications were: Gram-negative aerobe (0.6%), aerobic growth, referred (0.5%) and lactose-fermenter, referred (0.1%). Only six identifications were considered unacceptable; however, when combined with the susceptibility testing results, the response of E. coli, ESBL strain would also be considered incorrect (see below). The original source of this organism was a bacteremia occurring in a patient hospitalized in central Turkey (2009). Organism Identification E. coli is a Gram-negative facultative anaerobic member of the Enterobacteriaceae family. The morphology of E. coli often appears as a large colony, usually grey or white on blood containing agar with a smooth or slightly serrated edge, and is characteristically β-haemolytic. This species is oxidasenegative, displays lactose fermentation and grows well on MacConkey agar, a characteristic shared with Enterobacter spp. and Klebsiella spp. This species commonly produces shiny green metallic colonies on EMB agar. The odor of E. coli is unique, allowing for a rapid high quality preliminary identification when an indole test (i.e., spot indole-positive) is simultaneously performed. Production of indole from tryptophan is also seen with K. oxytoca, but other colonial morphology characteristics, usually differentiate between these two species. Most E. coli biotypes are motile by peritrichous flagella and ferment d-glucose which differentiates this species from Shigella spp., a phenotypically similar genus with nearly the same DNA-DNA hybridization and whole genome analysis when compared to E. coli. This species is easily identified using automated ID systems (Vitek 2, Microscan, BD Phoenix, etc).

2 E. coli are widely distributed in warm-blooded animals, ubiquitous in the environment, a beneficial colonizer of the human lower intestinal tract and has been an implicated pathogen involving virtually every human tissue and organ system. 1 E. coli are frequently commensals but may be opportunistic when conditions permit. Examples of permissive conditions are foreign body-associated infections (catheters), those in immunocompromised hosts, or where anatomic abnormalities (urinary blockage) may be present. Pathogenic E. coli are generally grouped into pathotypes depending on the disease they cause, the virulence factors they possess, and their host of isolation. 2-4 E. coli occur as either extraintestinal pathogenic E. coli (ExPEC) or intestinal or diarrheagenic E. coli. ExPEC strains carry genetic information that allow them to be pathogenic outside of the intestinal tract. There are six pathotypes of intestinal pathogenic E. coli. These are the enterotoxigenic E. coli (ETEC; watery diarrhea), enteroaggregative E. coli (EAEC; persistent diarrhea), enteroinvasive E. coli (EIEC; watery diarrhea, inflammatory colitis, or dysentery); enterohaemorrhagic E. coli (EHEC; nonbloody diarrhea, hemorrhagic colitis, or hemolytic-uremic syndrome); enteropathogenic E. coli (EPEC; nonbloody diarrhea), and the diffusely adherent E. coli (DEAC; nonbloody diarrhea). 3 There are two major pathotypes of extraintestinal strains: Urinary pathogenic E. coli (UPEC), and the meningitis-associated E. coli (MNEC) pathotypes. 3 UPEC strains (as in this sample ES-03) are the principle cause of urinary tract infections (UTI), 5 and are extremely common occurring in patients of all ages. In 2000, UTIs accounted for about 8.3 million outpatient visits. 6 Further, UTIs are the most common cause of nosocomial infections and Gram-negative sepsis in hospitalized patients. 6 Antimicrobial Susceptibility Testing (Ungraded) Participants were asked to perform antimicrobial susceptibility testing on this E. coli. This strain was selected to challenge proper identification and to determine antimicrobial susceptibility across numerous classes of agents. The initial reference laboratory antimicrobial susceptibility testing was conducted by standardized reference broth microdilution methods 7 and susceptibility was determined based on CLSI

3 document M100-S21 breakpoints. 8 The reference laboratory testing, reported a total of 27 agents (Table 1) that exhibited activity varying from high potency (16 drugs) to frank resistance (9 agents). Concensus accuracy criteria by susceptibility category are found in Table 2, for 28 antimicrobials having a significant sample of participant responses. Two antimicrobials (cefuroxime, piperacillin) did not achieve consensus ( 80%) due to MIC results at or within one doubling dilution step of intermediate breakpoints, see Table 1 and 2; furthermore, four agents (amoxicillin/clavulanate, ampicillin/sulbactam, cefoxitin, ticarcillin/clavulanate) required the combining of two susceptibility categories to achieve consensus. The acceptable performance by participants using the disk diffusion (DD) results 9 ranged from 33.3% (ticarcillin/clavulanate) to 100.0% (17 drugs). Lowest acceptable responses were for ticarcillin/clavulanate, cefazolin (64.7%), and piperacillin/tazobactam (75.0%), although these errors only represented 11 of 514 (2.1%) responses. Similarly, the MIC testing results 7, 8 had high levels of acceptable performance ranging from 87.6% (cefepime) to 100.0% (two drugs, with an overall rate of 97.4% for 11,418 tabulated values (see Table 2). The detection of the well defined resistance mechanisms in this strain was very acceptable, especially those directed against aminoglycosides ( % by MIC methods), fluoroquinolones ( %) and tetracycline (99.7%). The β-lactamase (OXA-1/30) present in this strain has a generally narrow affinity profile for older-generation penicillins and cephalosporins with key resistances to aminopenicillins (not associated with enzyme inhibitors), cephalothin (not cefazolin), and cefoxitin/cefuroxime (see Table 2). Clearly this organism is not an ESBL-producing strain because of having susceptible-level MIC values and zone diameters for screening monobactams (aztreonam) and cephems (cefotaxime, ceftazidime, ceftriaxone). A total of 106 participants (13.1%) indicated that this was an ESBL-strain; obviously an erroneous result that would result in a physician perception of clinical resistance that could limit the use of safe and less expensive treatment options among the β-lactams. Participants calling ES-03 (2011) an ESBL strain should critically review their susceptibility testing method/system for interpretive error.

4 Antimicrobial Therapy of UTIs There is wide variation in the choice of antimicrobial agents and duration of treatment for UTIs Reasons for treatment variations include concerns about bacterial resistance among uropathogens, drug availability and cost, expected efficacy and a desire to limit the effect of the antimicrobial on bacterial 12, 14 organisms that are not the target of therapy (so-called collateral damage). Recommendations for therapy for acute uncomplicated cystitis from the International Clinical Practice Guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women include nitrofurantoin (100 mg twice daily for five days), trimethoprim sulfamethoxazole (160/800 mg twice daily for three days), fosfomycin trometamol (3 g in a single dose), fluoroquinolones in three day regimens, and β-lactam agents in three to seven day regimens. 12 For acute pyelonephritis a urine culture is required and therapy should be tailored to the infecting uropathogen. 12 Due to concerns about trimethoprim sulfamethoxazole resistance there has been an increased use of fluoroquinolones which consequently has been accompanied by an increasing rate of fluoroquinolone 11, 13 resistance. The IDSA treatment guidelines recommend that trimethoprim sulfamethoxazole is an appropriate treatment in those regions or areas where resistance is no greater than 20%. 12 As specimens from patients with uncomplicated UTI infections frequently are not submitted for bacterial culture, prospective systematic active surveillance of urinary pathogens is needed to provide regional or 10, 14 local resistance prevalence information. Resistance Mechanisms Observed Wildtype E. coli isolates can be resistant to ampicillin, dominantly due to the presence of a plasmid-borne β-lactamase, TEM-1, but generally remain susceptible to penicillin/inhibitor combinations (e.g., amoxicillin-clavulanate and piperacillin/tazobactam). Increasingly, resistance to trimethoprimsulfamethoxazole (a commonly recommended agent for uncomplicated cystitis) is being recognized in E. coli, with a consequent increase in the prescription of the fluoroquinolone class of antimicrobial agents.

5 As a result, and also the wide-spread use of fluoroquinolones for respiratory tract infections, resistance to this class of agent among Enterobacteriaceae has escalated. All fluoroquinolones have the same mechanism of action, which consists of inhibition of the DNA gyrase and DNA topoisomerase IV bacterial enzymes leading to inhibition of DNA replication. 15 Usually resistance arises spontaneously due to point mutations that result in amino acid substitutions within DNA gyrase and DNA topoisomerase IV, often in combination with decreased expression of outer membrane porins and overexpression of multidrug efflux pump systems. 16 These nucleotide mutations are not transferable between species. However, plasmid-mediated quinolone resistance genes have been recognized in the last decade, and these determinants can be mobilized within Enterobacteriaceae. 17 DNA gyrase and DNA topoisomerase IV are large complex enzymes comprised of two pairs of subunits. The DNA gyrase subunits are GyrA and GyrB, while the corresponding units from DNA topoisomerase IV are ParC and ParE. 18 These two enzymes work simultaneously in the replication, transcription, recombination and repair of DNA. Quinolones interact with the DNA gyrase and DNA topoisomerase IV complexes resulting in conformational changes in the enzyme-bound DNA and enzyme itself, with subsequent release of lethal double-stranded DNA breaks. 19 DNA gyrase enzymes from Gram-negative bacteria are the main quinolone target site, while topoisomerase IV from Gram-positive is more prone to be inhibited by these antimicrobial agents. 19 Therefore, resistance mutations occur first in gyra in Gram-negative bacteria, as in Gram-positive organisms alterations occur first in parc. Usually, development of resistance among E. coli involves amino acid substitutions in a region of the GyrA or ParC subunit named the quinolone-resistance determining region (QRDR). This region overlaps with the DNA-binding surface of the enzyme, and within the E. coli DNA gyrase includes amino acids between positions 51 and 106, with positions 83 and 87 being considered as hot spots for mutation. 18

6 A first-step amino acid alteration within GyrA may be sufficient to confer high-level resistance to the older quinolone nalidixic acid. 16 However, additional mutations occurring in a step-wise manner are required for a high-level resistance phenotype to fluoroquinoles (this ES-03 [2011] isolate). In fact, single mutations in GyrA of E. coli clinical isolates increase the quinolone MIC results from 0.06 to 0.25 g/ml and first-step mutants are usually considered clinically susceptible ( 1 g/ml for ciprofloxacin susceptibility; CLSI criteria). A second alteration in GyrA or ParC is required for a clinically significant resistance phenotype (e.g. ciprofloxacin MIC, 4 g/ml). 18 Plasmid-mediated quinolone resistance (PMQR) has been increasingly reported in Enterobacteriaceae worldwide. 17 To date, three different transferable (fluoro)quinolone resistance mechanisms have been described: 1.) Five different qnr families, each with different numbers of alleles (qnra1 7, qnrb1 42, qnrc, qnrd, qnrs1 5) ( 2.) A modified aminoglycoside acetyl transferase gene [aac(6 )-1b-cr]; and 3.) a specific quinolone (qepa) and a multidrug resistance efflux pump (oqxab). Qnr proteins bind to DNA gyrase and DNA topoisomerase IV, and consequently protect these enzymes from being inhibited by fluoroquinolones. 18 The aminoglycoside acetyl transferase enzyme reduces the activity of ciprofloxacin due to N-acetylation at the amino nitrogen on its piperazinyl substituent. This enzyme also modifies gentamicin and tobramycin, but amikacin to a lesser extent. 20 The efflux pump systems decrease drug concentrations in the bacterial cells. 21 Although target site alterations are the most common and relevant fluoroquinolones resistance mechanisms, the AcrAB-TolC constitutive efflux pump system present in E. coli also plays a role. This system may become up-regulated decreasing drug uptake (decreased expression of outer membrane) and/or intracellular accumulation. 18 In addition, this intrinsic system can be induced by a variety of compounds and are often associated with other resistance mechanisms, such as target site alterations. The fluoroquinolone resistance mechanisms described above usually confers low-level resistance when present individually in a given clinical isolate. However, the fluoroquinolone resistance level increases as the resistance mechanisms and/or resistance associated-mutations accumulate.

7 The E. coli isolate recovered from the submitted specimen sample (ES-03 [2011]) was screened for the following fluoroquinolones resistance mechanisms: qnra-d, qnrs, aac(6 )-1b-cr and amino acid mutations in the DNA gyrase and DNA topoisomerase IV. The respective genes were amplified by PCR and sequenced. This E. coli clinical isolate was PCR-positive for aac(6 )-1b-cr and several mutations in the fluoroquinolone target sites were also observed, including S83L and D87N in GyrA, S80I in ParC and S458A in ParE. qnr genes were not detected. Alterations in S83 and D87 in GyrA and S80 in ParC have been previously associated with fluoroquinolones resistance and these are the most common modifications detected in clinical isolates of E. coli. 22 The S458 ParE alteration is located outside of the QRDR and appears to be associated with other mutations in GyrA and ParC. Therefore, the effect of this alteration in the fluoroquinolone MIC results still requires further investigation. 23 Other resistance detected by phenotypic (MIC breakpoints) or molecular methods were: aminoglycosides (gentamicin and tobramycin); tetracyclines and various older-generation β-lactams (aminopenicillins, carboxypenicillins, oral cephalosporins, cefoxitin) but not extended-spectrum β-lactams. Note that by the reported MIC results (Tables 1 and 2), this strain had susceptibility rates of: 93.1, 97.6, 95.9 and 95.1% for aztreonam, cefotaxime, ceftazidime and ceftriaxone, respectively. This E. coli does not qualify as an ESBL strain by CLSI screening critieria 7-9 or by ESBL PCR tests. Addendum February 2012 Following the analysis of the API sample ES-03 (2011), we discovered the significant occurrence of the response of "E. coli ESBL strain". The identification of E. coli was correct, but the categorization of the strain as an ESBL (extended-spectrum β-lactamase)-producing strain was an error. The strain did not have any of the phenotypic characteristics (MIC or zone diameter) of an ESBL (e.g., MICs of 2 µg/ml for cefotaxime, ceftriaxone, ceftazidime or aztreonam). In fact, the strain had susceptible MIC results to several older β-lactams including cefazolin, a first-generation cephalosporin. A questionnaire was sent to those laboratories reporting "E. coli ESBL strain" (112 sites) and we received replies from 62 laboratories (55%). The clear reason for the vast majority (96.7%) of the incorrect results was information generated by commercial susceptibility test systems (MIC endpoints) and one particular

8 method dominated (Vitek 2 with 85%). None of the participants made this diagnosis by using molecular methods to detect β-lactamase genes. Several users of the Vitek 2 system indicated that conflicting results were obtained (especially for cefepime) from the product and it's AES expert system produced erroneous modifications of MIC results (susceptible result changed to resistant) and the identification of an "ESBL-strain". This product obviously requires correction of the AES expert system or possibly expanded training of local laboratory personnel to interpret results. Thank you to the participating API subscribers for helping us establish the probable cause of this reporting error that could produce serious consequences to patient care and hospital epidemiology.

9 References 1. Manual of Clinical Microbiology, 10th edition. ASM Press, Washington D.C., Kaper JB. Pathogenic Escherichia coli. Int J Med Microbiol 2005; 295: Kaper JB, Nataro JP & Mobley HL. Pathogenic Escherichia coli. Nat Rev Microbiol 2004; 2: Johnson TJ & Nolan LK. Pathogenomics of the virulence plasmids of Escherichia coli. Microbiol Mol Biol Rev 2009; 73: Johnson JR & Russo TA. Molecular epidemiology of extraintestinal pathogenic (uropathogenic) Escherichia coli. Int J Med Microbiol 2005; 295: Clinical Infectious Disease. Cambridge University Press, New York, NY, Clinical and Laboratory Standards Institute. M07-A8. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard: eighth edition. CLSI, Wayne, PA, USA, Clinical and Laboratory Standards Institute. M100-S21. Performance standards for antimicrobial susceptibility testing: 21st informational supplement. CLSI, Wayne, PA, USA, Clinical and Laboratory Standards Institute. M02-A10. Performance standards for antimicrobial disk susceptibility tests; approved standard: tenth edition. CLSI, Wayne, PA, USA, Hillier S, Bell J, Heginbothom M et al. When do general practitioners request urine specimens for microbiology analysis? The applicability of antibiotic resistance surveillance based on routinely collected data. J Antimicrob Chemother 2006; 58: Colgan R, Johnson JR, Kuskowski M et al. Risk factors for trimethoprim-sulfamethoxazole resistance in patients with acute uncomplicated cystitis. Antimicrob Agents Chemother 2008; 52: 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: e

10 13. Sanchez GV, Master RN & Bordon J. Trimethoprim-sulfamethoxazole may no longer be acceptable for the treatment of acute uncomplicated cystitis in the United States. Clin Infect Dis 2011; 53: Gupta K, Hooton TM & Miller L. Managing uncomplicated urinary tract infection--making sense out of resistance data. Clin Infect Dis 2011; 53: Drlica K & Zhao X. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol Mol Biol Rev 1997; 61: Hopkins KL, Davies RH & Threlfall EJ. Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments. Int J Antimicrob Agents 2005; 25: Veldman K, Cavaco LM, Mevius D et al. International collaborative study on the occurrence of plasmid-mediated quinolone resistance in Salmonella enterica and Escherichia coli isolated from animals, humans, food and the environment in 13 European countries. J Antimicrob Chemother 2011; 66: Jacoby GA. Mechanisms of resistance to quinolones. Clin Infect Dis 2005; 41 Suppl 2: S120-S Hooper DC. Mechanisms of fluoroquinolone resistance. Drug Resist Updat 1999; 2: Robicsek A, Strahilevitz J, Jacoby GA et al. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 2006; 12: Yamane K, Wachino J, Suzuki S et al. New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate. Antimicrob Agents Chemother 2007; 51: Bansal S & Tandon V. Contribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates. Int J Antimicrob Agents 2011; 37: Sorlozano A, Gutierrez J, Jimenez A et al. Contribution of a new mutation in pare to quinolone resistance in extended-spectrum-beta-lactamase-producing Escherichia coli isolates. J Clin Microbiol 2007; 45:

11 Table 1. Listing of expected susceptibility category results for the E. coli strain found in ES-03 (2011). Antimicrobial agents by susceptibility category (reference MIC in µg/ml) a Susceptible Intermediate Resistant Aztreonam (0.25) b Cefuroxime (16) Ampicillin (>8) Cefepime (1) Piperacillin (32) Ampicillin/Sulbactam (32/16) Cefoperazone (4) Amoxicillin/Clavulanate (>8/4) Cefoperazone/Sulbactam (2/2) Cefoxitin (>16) Ceftazidime (0.25) b Ciprofloxacin (>4) Ceftriaxone (0.12) b Levofloxacin (>4) Piperacillin/Tazobactam (8/4) Gentamicin (>8) Doripenem ( 0.06) Tobramycin (>16) Ertapenem ( 0.06) Tetracycline (>8) Imipenem ( 0.12) Meropenem ( 0.06) Amikacin (4) Colistin ( 0.25) Minocycline (1) Tigecycline (0.12) TMP/SMX ( 0.5/9.5) c a. Categories assigned per CLSI M07-A8 and M100-S21 (2011) criteria 8 or USA-FDA approved product package insert (tigecycline). b. Broad-spectrum or "extended-spectrum" agents used to screen E. coli, Klebsiella spp or P. mirabilis for ESBL enzymes; all were negative (MIC, 1 µg/ml) 8, not requiring further confirmational tests. c. TMP/SMX = trimethoprim/sulfamethoxazole

12 Table 2. Participant performance for selected agents ( 10 responses by one or both test methods) listed by agar disk diffusion (DD) and quantitative MIC methods for ES-03 (2011), an E. coli strain with resistance to fluoroquinolones and selected other agents. DD MIC Antimicrobial Agent Acceptable susceptibility category No. % correct a No. % correct a Amikacin Susceptible Amoxicillin/Clavulanate Intermediate-Resistant a Ampicillin Resistant Ampicillin/Sulbactam Intermediate-Resistant a Aztreonam b Susceptible Cefazolin Susceptible Cefepime Susceptible Cefotaxime b Susceptible Cefoxitin Intermediate-Resistant a Ceftazidime b Susceptible Ceftriaxone b Susceptible Cefuroxime - c Cephalothin Resistant Ciprofloxacin Resistant Doripenem Susceptible Ertapenem Susceptible Gentamicin Resistant Imipenem Susceptible Levofloxacin Resistant Meropenem Susceptible Nitrofurantoin d Susceptible Norfloxacin d Resistant Piperacillin - c Piperacillin/Tazobactam Susceptible Tetracycline Resistant Ticarcillin/Clavulanate Susceptible-Intermediate a Tigecycline Susceptible Tobramycin Resistant Trimethoprim d Susceptible TMP/SMX Susceptible a. Proportion (%) conforming to 80% consensus of all participant s results and in agreement with reference laboratory MIC results interpreted by CLSI M100-S21 breakpoints 8. The combining of two categories to achieve 80% was required for three agents. b. Agents used to detect ESBL enzyme-producing strains of E. coli, all having results in the susceptible, screen-negative range. c. - = intermediate level of activity with categorized results 8 ranging from susceptible to resistant (see Table 1 for reference MIC value); no acceptable category was assigned. d. Agents used clinically for UTI only, each showing high rates of susceptibility ( %) by both methods, except norfloxacin.