1. 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
CTX-M-1 + - 8
0.125 2
0.016 3
0.023
2.106 24
0.032
1.5 32
0.25 12
CTX-M-1 + TEM-1 4 48
0.094 16
0.19 6
0.064
CTX-M-3 + SHV-12
0.047
CTX-M-14 1
6.107
CTX-M-14 + TEM-1
0.75
CTX-M-15
>256
192 96 64
128
CTX-M-15 + TEM-1
6.108
4.107
0.38 NI
9.108
CTX-M-27 + TEM-1
TEM-24
K. pneumoniae
CTX-M-15 + SHV-1
CTX-M-15 + SHV-1 + TEM-1
0.5
C. freundii
CTX-M-3
C. koseri
E. cloacae
5.107
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 g
Centrifugation
2 min at 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