N°XXXX Rapid detection of extended-spectrum BETA-lactamase producing Enterobacteriaceae from urine using the ESBL NDP test Patrice Nordmann, Laurent Poirel, and Laurent Dortet  University of Fribourg, Fribourg, Switzerland (L. Poirel, P. Nordmann) Hopital Fribourgeois-hôpital Cantonal, Fribourg, Switzerland (P. Nordmann) INSERM U914, K.-Bicêtre, France (L. Dortet, L. Poirel, P. Nordmann) INTRODUCTION METHODS RESULTS Multidrug resistance is now emerging worldwide at an alarming rate among Gram negatives, causing both community-acquired and nosocomial infections. One of the most important emerging resistance traits corresponds to resistance to broad-spectrum ß-lactams in Enterobacteriaceae, which is mainly associated to production of clavulanic acid inhibited extended-spectrum ß-lactamases (ESBLs). A variety of ESBLs have been reported in Enterobacteriaceae, being mostly of the CTX-M-, TEM- and SHV-types. ESBL producers are mostly Escherichia coli and Klebsiella pneumoniae, being the main source of community- and hospital-acquired infections. In addition, those bacteria are responsible for divers types of infection among which urinary tract infections are predominant. This detection is necessary in order to screen patients, and subsequently improve hospital infection control practices, to curb inappropriate antibiotic use, and therefore to prolong the efficacy of the currently available antibiotics. Recently, the ESBL NP test has been developed for a rapid identification of ESBL-producing Enterobacteriaceae. This technique, based on the detection of hydrolysis of the β-lactam ring of a 3rd generation cephalosporin, cefotaxime, is rapid, sensitive and specific. From June to September 2012, the ESBL NDP test was performed on 450 urine samples recovered from patients hospitalized in a tertiary care center. Urine specimens with more than 104 leucocytes/ml and positive for Gram-negative bacilli after Gram staining were included in the study. Results of the ESBL NDP test were compared to those of the antibiogram and the double disk diffusion method. Subsequently, all ESBL producers were characterized phenotypically (MIC determination) and at their molecular level. ESBL NDP test on urine samples (n=450) Figure 1. Design and results of the study All ESBL-E were resistant to cefotaxime (the substrate used in the ESBL NDP test) except a single E. coli isolate producing TEM-24 (Table 1). These isolates were also resistant to other non-β-lactam antibiotics used for treating UTIs such as gentamicin (64.7%), co-trimoxazole (83.3%), fosfomycin (27.5%), ciprofloxacin (83.3%), amoxicillin + clavulanate (29.4%), piperacillin + tazobactam (13.7%), cefoxitin (15.7%), nitrofurantoin (9.8%), and remained susceptible to all carbapenems (imipenem, ertapenem, meropenem) and to temocillin. NI _ + n = 49 n = 6 n = 395 UriSelect™ 4 UriSelect™ 4 UriSelect™ 4 ESBL NDP test on grown colonies ESBL NDP test on grown colonies ESBL NDP test on grown colonies ESBL NP test Principle Protocol Interpretation after 20 min _ _ _ Acid production + + + n = 48 n = 1 n = 2 n = 0 n = 4 n = 395 H2N R COOH R’ O HO S pH Non-ESBL Non-ESBL Non-ESBL Non-ESBL Non-ESBL Non-ESBL ESBL ESBL ESBL ESBL ESBL ESBL 3rd generation cephalosporin n = 48 n = 0 n = 0 n = 1 n = 2 n = 0 n = 0 n = 4 n = 0 n = 0 n = 1 n = 394 N O R S COOH R’ Colorimetric detection OXA-1 producing E. coli TEM-24-producing E. coli ESBL Table 1. Characterization of the ESBL producers N O R S COOH R’ Species CFU / ml β-Lactamase content ESBL NDP test on urine ESBL NDP test on colonies MICs (μg/ml) CTX CTX + TZB CAZ CAZ + TZB FEP FEP + TZB E. coli 1.2.106 CTX-M-1 + - 8 0.125 2 0.016 3 0.023 2.106 24 0.032 1.5 3.8.107 32 0.25 12 2.8.107 CTX-M-1 + TEM-1 4 48 0.094 16 1.1.107 0.19 6 0.064 CTX-M-3 + SHV-12 0.047 9.4.107 CTX-M-14 1 2.8.108 6.107 CTX-M-14 + TEM-1 0.75 1.3.106 CTX-M-15 >256 3.3.107 9.8.107 192 5.2.108 4.1.107 2.3.108 96 6.5.107 5.3.107 9.4.106 1.6.107 1.2.108 64 1.8.107 128 9.5.106 CTX-M-15 + TEM-1 2.1.108 6.3.107 6.108 4.107 6.2.108 0.38 3.4.107 NI 9.108 CTX-M-27 + TEM-1 8.9.106 TEM-24 K. pneumoniae 3.1.106 1.1.108 CTX-M-15 + SHV-1 CTX-M-15 + SHV-1 + TEM-1 4.3.108 0.5 3.7.107 2.7.108 1.3.108 4.3.107 3.9.108 3.1.107 C. freundii CTX-M-3 C. koseri 2.1.107 E. cloacae 5.1.107 5.107 1.5.108 1.4.108 SHV-12 + TEM-1 +/- 0.119 (cefotaxime) + tazobactam Stable pH A B C 100 μl B-PER II lysis buffer A B C 0.5 ml H2O A B C 1.5 ml Urine Remove the supernatant Remove the supernatant Characteristics of the ESBL NDP test directly performed on clinical urine samples Sensitivity 98.0 % Specificity 99.8 % Positive predictive value Negative predictive value Centrifugation 2 min at 12 000 g Centrifugation 2 min at 12 000 g A B C 100 μl PR solution + cefotaxime (at 6 mg/ml) A B C 100 μl PR solution 10 μl Tazobactam (at 40 mg/ml) CONCLUSIONS PURPOSE Using the ESBL NDP test directly on urine led to detect all ESBL producers except two that were susceptible to cefotaxime and two that were found in not significative concentration in the urine sample. Only 1.3% of non interpretable results were obtained. A B C Evaluate the ability of the ESBL NP test to detect ESBL producers among Enterobacteriaceae directly from urine samples. Overexpressed cephalosporinase+/- ESBL Not interpretable ESBL No BLSE A B C A B C A B C A B C