Abstract
With the β-Lacta test, production of extended-spectrum β-lactamases (ESBLs) was assayed in 200 urine samples showing Gram-negative bacilli during direct microscopic examination. While 168 samples tested negative, all samples yielding ESBL-producing Enterobacteriaceae after culture gave positive (n = 30) or uninterpretable (n = 2) results. The sensitivity and specificity of ESBL detection were 94% and 100%, respectively.
TEXT
A survey conducted from 2002 to 2010 by the French national infection control program demonstrated an increase of 282% in the incidence of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBLE), particularly ESBL-producing Escherichia coli (1). The increasing importance of multiresistant ESBL-producing E. coli strains in the community should make clinicians aware of potential treatment failures associated with serious and potentially life-threatening infections (2). A recent study by Peralta et al., conducted in 19 Spanish hospitals, found that the ESBLE causing bacteremia were mainly from the urinary (55.3%) and biliary (12.7%) tracts. E. coli accounted for 89% of all ESBLE strains, and 45.7% of these were multidrug resistant (e.g., resistant to β-lactam–β-lactamase inhibitor combinations, cephalosporins, quinolones, and aminoglycosides) (3). Furthermore, empirical antibiotic treatment was adequate in only 48.8% of the cases, and the in-hospital mortality was 20.9% (3). ESBL-producing E. coli isolates, particularly those producing CTX-M enzymes, account for a significant number of cases of bacteremia in hospitalized and nonhospitalized patients (4). Moreover, Livermore has reported that, since 2003, 90% of ESBL-producing E. coli isolates in the United Kingdom produce CTX-M-15 (5).
Urinary tract infections (UTI) are among the most frequent bacterial infections in the community and in the health care setting (6). Broad-spectrum cephalosporins, β-lactam–β-lactamase inhibitor combinations, or fluoroquinolones are recommended for first-line empirical therapy of sepsis arising from the urinary tract in community-acquired and nosocomially acquired cases (4, 7). These recommendations might be difficult to apply if a significant proportion of such infections is caused by ESBLE (4).
A new chromogenic test (β-Lacta test [BLT]; Bio-Rad, Marnes-La-Coquette, France) was developed for the rapid detection (in less than 15 min) of strains of Enterobacteriaceae with decreased susceptibility or resistance to third-generation cephalosporins (3GC) conferred by enzymes such as ESBLs and carbapenemases. This test provides useful information to guide antibiotic treatment before full results of antimicrobiotic susceptibility testing are available (8). The aim of this study was to highlight the time saved in cases of UTI when the BLT is performed directly with urine rather than bacterial colonies.
Urine samples.
All urine samples received over a 3-month period and showing Gram-negative bacilli (GNB) upon direct microscopic examination and on Gram stains of uncentrifuged urine were prospectively included. Urine samples with hematuria which would interfere with the chromogenic BLT were excluded.
β-Lacta test.
The BLT was performed directly on urine sediments according to the manufacturer's recommendations (8). Two collection periods were scheduled to test for possible variations in BLT efficiency; during the first 5 weeks, 1-ml urine samples collected from 100 patients were centrifuged for 2 min at 3,000 × g in Eppendorf tubes, and during the following 7 weeks, 1.5-ml samples collected from an additional 100 patients were centrifuged for 5 min, also at 3,000 × g. After elimination of the supernatant, the BLT was performed in the sediment-containing tube. The sediment was completely homogenized in one drop (ca. 50 μl) of each reagent, and after incubation for 15 min at room temperature, the test was interpreted visually as follows: no change in color indicated a negative result, a color change to red or purple indicated a positive result, and a color change to orange was uninterpretable. In some cases, an immediate color change was observed (in less than 1 min).
Antibiotic susceptibility testing.
Standard disk diffusion results were interpreted 48 h after the BLT, and 3GC (i.e., cefotaxime or ceftazidime)-resistant isolates were screened for ESBL production using the double-disk synergy test by following the recommendations of the Comité de l'Antibiogramme of the Société Française de Microbiologie (CA-SFM) (9).
Molecular characterization of β-lactamase genes.
All strains suspected of producing one or more acquired broad-spectrum β-lactamases (BSBLs) or extended-spectrum β-lactamases (ESBLs) and strains resistant to ceftazidime or cefotaxime were screened using PCR and sequencing as described previously (10, 11).
Culture characteristics of GNB.
In total, 200 urine samples containing GNB, as seen using direct microscopy and Gram staining of uncentrifuged urine, were included in this study. From these, 221 strains grew on Uriselect 4 agar (Bio-Rad) after 16 to 24 h of incubation at a threshold of detection of ≥104 CFU/ml. They were mainly Enterobacteriaceae (n = 207; 94%), including 147 E. coli strains (71%), 23 Klebsiella pneumoniae strains (11%), 21 Enterobacter species strains (10%), 9 Proteus species strains (4%), 4 Morganella morganii strains, and 3 Citrobacter koseri strains (Table 1). Fourteen obligate aerobes (6%) were recovered on the same medium, including 10 Pseudomonas species strains, 3 Stenotrophomonas maltophilia strains, and 1 Acinetobacter baumannii strain (Table 1).
TABLE 1.
Phenotypes of Gram-negative bacilli isolated from urine samples subjected to the β-Lacta test
| Organism(s) or test result | No. of strains with indicated phenotype |
Total | |||
|---|---|---|---|---|---|
| Wild | BSBLa | ESBL | AmpC-type β-lactamasesb | ||
| Escherichia coli | 79 | 48 | 16 | 4 | 147 |
| Klebsiella pneumoniae | 9 | 0 | 14 | 0 | 23 |
| Enterobacter spp. | 13 | 0 | 3 | 5 | 21 |
| Proteus mirabilis | 3 | 4 | 0 | 1 | 8 |
| Citrobacter koseri | 3 | 0 | 0 | 0 | 3 |
| Morganella morganii | 3 | 1 | 0 | 0 | 4 |
| Proteus vulgaris | 1 | 0 | 0 | 0 | 1 |
| Obligate aerobesc | 9 | 0 | 0 | 5 | 14 |
| Total | 120 | 53 | 33 | 15 | 221 |
| No. β-Lacta test positive | 0 | 0 | 31 | 0 | 31 |
| No. β-Lacta test uninterpretable | 0 | 0 | 2 | 0 | 0 |
BSBL, broad-spectrum β-lactamases, including TEM-1, TEM-2, OXA-1, and IRT-2.
AmpC-type β-lactamases conferring resistance to ceftazidime or cefotaxime (chromosome or plasmid mediated).
Ten Pseudomonas aeruginosa strains, one Acinetobacter baumannii strain, and three Stenotrophomonas maltophilia strains.
Performance of the BLT.
In total, 30 (15%) of the 200 urine samples were found to be BLT positive, and two (1%) gave a uninterpretable result. During the first collection period, 10 out of 100 samples tested positive with the BLT and two samples gave uninterpretable results (Table 2). During the second collection period, BLT seemed to be somewhat more efficient, with 20 samples out of 100 testing positive and all results being interpretable (Table 2).
TABLE 2.
β-Lacta test results during the first and second collection periods for extended-spectrum β-lactamase-producing Enterobacteriaceae
| β-Lacta sample collection period | ESBLE | Amt of GNB at microscopic examination | CFU/ml by culture | Initial color | Immediate color change (<1 min) | Color at 15 min | Result | bla gene |
|---|---|---|---|---|---|---|---|---|
| 1st | Klebsiella pneumoniae | Several | >105 and 105 P. aeruginosa | Yellow | Yes | Purple | Positive | CTX-M-15 |
| Klebsiella pneumoniae | Plenty | 105 | Yellow | No | Orange | Uninterpretable | CTX-M-15 | |
| Escherichia coli | Abundant | 105 | Yellow | No | Red | Positive | CTX-M-15 | |
| Klebsiella pneumoniae | Abundant | >105 and >105 E. coli | Yellow | No | Red | Positive | CTX-M-15 | |
| Escherichia coli | Few | >105 | Yellow | No | Red | Positive | CTX-M-64* | |
| Klebsiella pneumoniae | Several | >105 | Yellow | No | Red | Positive | CTX-M-15 | |
| Klebsiella pneumoniae | Plenty | 105 | Yellow | Yes | Purple | Positive | CTX-M-15 | |
| Escherichia coli | Abundant | 105 | Yellow | No | Red | Positive | CTX-M-15 | |
| Escherichia coli | Plenty | >105 | Yellow | No | Red | Positive | CTX-M-55 | |
| Klebsiella pneumoniae | Several | >105 | Yellow | No | Purple | Positive | CTX-M-15 | |
| Escherichia coli | Few | 104 | Yellow | Yes | Orange | Uninterpretable | CTX-M-1 | |
| Klebsiella pneumoniae | Plenty | >105 | Yellow | No | Purple | Positive | CTX-M-15 | |
| 2nd | Enterobacter cloacae | Abundant | >105 | Orange | Yes | Purple | Positive | CTX-M-15 |
| Escherichia coli | Abundant | 105 | Yellow | Yes | Purple | Positive | CTX-M-55 | |
| Enterobacter cloacae | Few | 105 | Yellow | No | Purple | Positive | CTX-M-15 | |
| Klebsiella pneumoniae | Abundant | >105 | Yellow | Yes | Purple | Positive | CTX-M-15 | |
| Klebsiella pneumoniae | Several | >105 | Yellow | No | Purple | Positive | CTX-M-15 | |
| Klebsiella pneumoniae | Few | >105 | Yellow | No | Purple | Positive | CTX-M-14 | |
| Escherichia coli | Several | >105 and >105 Enterococcus species | Yellow | No | Purple | Positive | CTX-M-15 | |
| Escherichia coli | Abundant | >105 | Yellow | No | Purple | Positive | CTX-M-15 | |
| Escherichia coli | Several | 105 | Yellow | No | Purple | Positive | CTX-M-55 | |
| Escherichia coli | Several | 105 | Yellow | Yes | Purple | Positive | CTX-M-15 | |
| Escherichia coli | Few | >105 and >105 E. cloacae | Yellow | No | Red | Positive | CTX-M-64**a | |
| Escherichia coli | Abundant | 105 | Yellow | No | Purple | Positive | CTX-M-15 | |
| Escherichia coli | Several | 105 | Yellow | No | Red | Positive | CTX-M-14 | |
| Escherichia coli, Klebsiella pneumoniae | Few | 105, 105, and 105 A. baumannii | Yellow | No | Red | Positive | CTX-M-15 | |
| Klebsiella pneumoniae | Abundant | 105 | Yellow | No | Red | Positive | CTX-M-27 | |
| Escherichia coli | Plenty | >105 | Yellow | No | Red | Positive | CTX-M-1 | |
| Escherichia coli | Several | >105 | Yellow | Yes | Purple | Positive | CTX-M-55 | |
| Klebsiella pneumoniae | Plenty | >105 | Yellow | No | Purple | Positive | CTX-M-15 | |
| Enterobacter cloacae | Several | 105 | Yellow | Yes | Purple | Positive | CTX-M-15 | |
| Klebsiella pneumoniae | Several | >105 | Yellow | No | Purple | Positive | CTX-M-15 |
CTX-M-64** was found twice in the same patient in two independently collected urine samples.
Phenotypic characterization of all GNB isolates from urine (Table 1) confirmed that samples giving a positive or an uninterpretable BLT result were ESBL producers, and no negative BLT results were obtained for ESBL producers. All GNB strains producing β-lactamases other than ESBLs, such as broad-spectrum β-lactamases (TEM-1, TEM-2, IRT-2, and OXA-1) and AmpC-type enzymes, gave negative BLT results. Thirty-three ESBLs were isolated from the 32 BLT-positive urine samples, including 16 E. coli, 14 K. pneumoniae, and 3 Enterobacter species strains (Table 1). BLT has shown sensitivities of 87% in the first collection period and of 100% in the second and a specificity of 100% in both periods. The presence of other bacterial morphotypes in the polymorphic flora seen at direct microscopic examination did not interfere with the BLT results.
The molecular results confirmed the phenotypic enzyme characterizations and revealed that all ESBLs belonged to the CTX-M group (Table 2), in apparent keeping with the ongoing CTX-M β-lactamase pandemic (5, 12).
The test yielded very high values for both sensitivity (94%) and specificity (100%). Recently, Renvoisé et al., analyzing isolates grown for 16 to 24 h, demonstrated that the sensitivity and specificity of BLT were 99.6% and 87.7%, respectively, for Enterobacteriaceae overexpressing ampC and that both were 100% for ESBL producers (8). This demonstrates that the β-lactam ring of the BLT chromogenic substrate (HMRZ-86) is very efficiently hydrolyzed by ESBL but not AmpC-type activities.
All screening tests for rapid detection of ESBL-producing GNB require at least 16 to 24 h, including those that use specific agar media, e.g., ESBL agar (AES Chemunex), ChromID ESBL agar (bioMérieux), or Brilliance ESBL agar (Oxoid). They have sensitivities and specificities of 81.3 to 87.5% and 60.8 to 82.1%, respectively (13). Recently, a biochemical test, ESBL NDP, was proposed by Nordmann et al. (14). Preliminary culturing of the isolate was required, but the test took less than 1 h; it was found to have a specificity of 100% and a sensitivity of 92.6%.
In conclusion, BLT may be considered an efficient test for the detection of ESBL in urine, and due to the rapidity and ease with which it is performed, it is a valuable adjunct in specifying empirical UTI management, including measures to limit cross-transmission. The BLT may be an adequate tool for efficient detection of carbapenemase-producing Enterobacteriaceae in countries with an elevated prevalence of theses enzymes (e.g., Klebsiella pneumoniae carbapenemase and class B carbapenemases) (15).
ACKNOWLEDGMENTS
We thank Manette Juvin and Caroline Dallenne for support during this study.
This study was conducted as part of our routine work, and we received no extra funding.
We have no conflicts of interest to declare.
Footnotes
Published ahead of print 30 July 2014
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