Rapid Detection of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae

Enterobacterial strains producing clavulanic-acid-inhibited extended-spectrum β-lactamases (ESBLs) are increasingly reported worldwide. Conventional detection of ESBL production remains time-consuming (24 to 48 h). Therefore, the ESBL NDP (Nordmann/Dortet/Poirel) test was developed for a rapid identification of ESBLs in Enterobacteriaceae. This biochemical test was based on the in vitro detection of a cephalosporin (cefotaxime) hydrolysis that is inhibited by tazobactam addition. The ESBL activity was evidenced by a color change (red to yellow) of a pH indicator (red phenol) due to carboxyl-acid formation resulting from cefotaxime hydrolysis that was reversed by addition of tazobactam (positive test). The ESBL NDP test was applied to cultured strains (215 ESBL producers and 40 ESBL nonproducers). Its sensitivity and specificity were 92.6% and 100%, respectively. Its sensitivity (100%) was excellent for detection of CTX-M producers. A few ESBL producers (n = 16) that remained susceptible to cefotaxime were not detected. The test was also evaluated on spiked blood cultures and showed excellent sensitivity and specificity (100% for both). The test was rapid (less than 1 h) and cost-effective. It can be implemented in any health care facility and is well adapted for infection control purposes in particular.     

Rapid Detection of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae from Urine Samples by Use of the ESBL NDP Test

From June to September 2012, 500 urine samples were recovered from patients with urinary tract infections (UTI) due to Gram-negative bacilli (≥10⁴ leukocytes/ml and ≥10⁵ Gram-negative isolates/ml) who visited the University hospital Bicêtre (France). They were challenged with extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) using the rapid diagnostic ESBL NDP test. Results of the ESBL NDP test were compared to the results of the double-disc susceptibility test (DDST) performed on solid-agar plates and molecular identification of the β-lactamase genes. Among the 450 nonduplicate urine samples, 11.3% were positive for ESBL-E by using the DDST, the ESBL determinants being mostly of the CTX-M type (CTX-M-15) according to molecular testing. Results of the ESBL NDP test were obtained within 15 min. The sensitivity and specificity of the ESBL NDP test were 98% and 99.8%, respectively, whereas the positive and negative predictive values of this test were 98% and 99.8%, respectively. A perfect correlation between cefotaxime resistance and positivity of the ESBL NDP test was observed. Therefore, the ESBL NDP test offers a powerful tool for a rapid identification of ESBL-E and associated resistance to expanded-spectrum cephalosporins. It may be useful in particular for guiding first-line antibiotic therapy.     

The β-Lacta Test for Direct Detection of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae in Urine

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.     

Evaluation of Double-Disk Potentiation and Disk Potentiation Tests Using Dipicolinic Acid for Detection of Metallo-β-Lactamase-Producing Pseudomonas spp. and Acinetobacter spp

Accurate detection of metallo-β-lactamase (MBL)-producing Pseudomonas spp. and Acinetobacter spp. became very important with the increasing prevalence of carbapenem-nonsusceptible clinical isolates. The performance of phenotypic MBL detection methods may depend on the types of MBL and the characteristics of the isolates. A high false-positive rate is a problem with EDTA-based MBL detection methods. We evaluated the performance of double-disk potentiation tests (DDPTs) and disk potentiation tests (DPTs) with dipicolinic acid (DPA) using 44 isolates of Pseudomonas spp. and Acinetobacter spp. producing IMP-1-like, VIM-2-like, and SIM-1 type MBLs. Also, we characterized P. aeruginosa isolates with positive imipenem (IPM)-DPA DDPT, but negative meropenem (MEM)-DPA DDPT, and determined possibility of improving a DDPT by using MacConkey agar. Among five different DDPT methods, the IPM-DPA 250-μg method showed the highest sensitivity (97.7%) and specificity (100%). Among four DPT tests, the highest sensitivity (100%) was shown by the IPM-EDTA 1,900-μg disk method, but the specificity was very low (11.4%). Five of six P. aeruginosa isolates with false-negative DDPTs with MEM-DPA 250-μg disks carried bla(IMP-6,) and the high level resistance to MEM (MIC ≥ 512 μg/ml) was reduced by the presence of phenylalanine arginine β-naphtylamide. Improvement of DDPTs was observed when MacConkey agar was used instead of Mueller-Hinton agar. In conclusion, DPA is a better MBL inhibitor than EDTA for detection of Pseudomonas spp. and Acinetobacter spp. with IMP-1-like, VIM-2-like, and SIM-1-type MBLs. In DPA DDPTs, IPM disks perform better than MEM disks when the isolates are highly resistant to MEM due to the overexpression of efflux pumps.     

Prevalence,Risk Factors,and Transmission Dynamics of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae: a National Survey of Cattle Farms in Israel in 2013

Our objectives were to study the prevalence, risk factors for carriage, and transmission dynamics of extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae (ESBLPE) in a national survey of cattle. This was a point prevalence study conducted from July to October 2013 in Israel. Stool samples were collected from 1,226 cows in 123 sections on 40 farms of all production types. ESBLPE were identified in 291 samples (23.7%): 287 contained Escherichia coli and 4 contained Klebsiella pneumoniae. The number of ESBLPE-positive cows was the highest in quarantine stations and on fattening farms and was the lowest on pasture farms (P = 0.03). The number of ESBLPE-positive cows was the lowest in sections containing adult cows (age, >25 months) and highest in sections containing calves (age, <4 months) (P < 0.001). Infrastructure variables that were significant risk factors for ESBLPE carriage included crowding, a lack of manure cleaning, and a lack of a cooling (P < 0.001 for each), all of which were more common in sections containing calves. Antimicrobial prophylaxis was given almost exclusively to calves and was associated with a high number of ESBLPE carriers (P < 0.001). The 287 E. coli isolates were typed into 106 repetitive extragenic palindromic (REP)-PCR types and mostly harbored bla_CTX-M-1 or bla_CTX-M-9 group genes. The isolates on the six farms with ≥15 isolates of ESBLPE were of 4 to 7 different REP-PCR types, with one dominant type being harbored by about half of the isolates. Fourteen types were identified on more than one farm, with only six of the farms being adjacent to each other. The prevalence of ESBLPE carriage is high in calves in cowsheds where the use of antimicrobial prophylaxis is common. ESBLPE disseminate within cowsheds mainly by clonal spread, with limited intercowshed transmission occurring.     

Detection of Extended-Spectrum-β-Lactamase-Producing Escherichia coli ST131 and ST405 Clonal Groups by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Escherichia coli sequence type 131 (ST131) and ST405 are important clonal groups, because they are associated with the global increase of extended-spectrum-β-lactamase (ESBL) producers. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is emerging as a rapid, inexpensive, and accurate method for bacterial identification. We investigated the detection performance of MALDI-TOF for the ST131 and ST405 clonal groups using 41 ST131-O25b, 26 ST131-O16, and 41 ST405 ESBL-producing isolates and 41 ESBL-producing isolates frrom other STs. The main spectra representing each clonal group were used for classification with Biotyper (Bruker Daltonics GmbH, Bremen, Germany). The peak that had the highest area under the receiver-operating characteristic curve generated by ClinProTools (Bruker) was detected with FlexAnalysis (Bruker), and an optimal signal-to-noise ratio cutoff was determined. The optimal detection models were generated by ClinProTools. Classification by Biotyper could detect the ST131-whole (O25b and O16 together) group with a sensitivity of 98.5% and a specificity of 93.9%. With FlexAnalysis, a peak of 9,720 Da detected the ST131-whole group with a sensitivity of 97.0% and a specificity of 91.5% at a cutoff value of 8.0. The ClinProTools models exhibited good performance for the detection of the ST131-whole group (sensitivity and specificity, 94.0% and 92.7%, respectively), the ST131-O25b group (95.1% and 98.2%, respectively), and the ST405 group (90.2% and 96.3%, respectively). MALDI-TOF MS had high detection performance for the ST131-whole, ST131-O25b, and ST405 clonal groups. MALDI-TOF MS should be considered as an alternative method to monitor the epidemiology of the ESBL-producing E. coli ST131 and ST405 clonal groups.     

First Description of an Extended-Spectrum-β-Lactamase-Producing Multidrug-Resistant Escherichia fergusonii Strain in a Patient with Cystitis

Extended-spectrum-β-lactamase (ESBL)-producing organisms have captured the attention of clinicians and laboratorians and are agents of nosocomial and community onset infections (J. D. Pitout and K. B. Laupland, Lancet Infect. Dis. 8:159-166, 2008). ESBLs in many enterobacteriaceae and in nonfermenting Gram-negative organisms have been described (K. Bush and G. A. Jacoby, Antimicrob. Agents Chemother. 54:969-976, 2010). We present the first case of a clinical isolate of multidrug-resistant Escherichia fergusonii expressing an extended-spectrum-β-lactamase (ESBL).     

Rapid Detection of OXA-48-Producing Enterobacteriaceae by Matrix-Assisted Laser Desorption Ionization?Time of Flight Mass Spectrometry

A rapid and sensitive (100%) matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect OXA-48-type producers, using 161 previously characterized clinical isolates. Ertapenem was monitored to detect carbapenem resistance, and temocillin was included in the assay as a marker for OXA-48-producers. Structural analysis of temocillin is described. Data are obtained within 60 min.     

Rapid Detection of β-Lactamase-Hydrolyzing Extended-Spectrum Cephalosporins in Enterobacteriaceae by Use of the New Chromogenic βLacta Test

The chromogenic βLacta test developed for the rapid detection of β-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae revealed good performance with extended-spectrum β-lactamase (ESBL) producers (97.5% true-positive results). However, false-negative results occurred with chromosomal AmpC hyperproducers and plasmid AmpC producers, whereas uninterpretable results were mostly due to VIM-1 carbapenemase producers and possibly low levels of expressed ESBLs.     

Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Rapid Detection of β-Lactam Resistance in Enterobacteriaceae Derived from Blood Cultures

The identification of pathogens directly from blood cultures by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be a valuable tool for improving the treatment of patients with sepsis and bacteremia. However, the increasing incidence of multidrug-resistant Gram-negative bacteria makes it difficult to predict resistance patterns based only on pathogen identification. Most therapy regimens for sepsis caused by Gram-negative rods consist of at least one β-lactam antibiotic. Thus, it would be of great benefit to have an early marker of resistance against these drugs. In the current study, we tested 100 consecutive blood cultures containing Enterobacteriaceae for resistance against 3rd-generation cephalosporins in a MALDI-TOF MS β-lactamase assay. Escherichia coli was also tested for resistance against aminopenicillins. The results of the β-lactamase assay were compared with those of conventional methods. The assay permitted discrimination between E. coli strains that were resistant or susceptible to aminopenicillins with a sensitivity and a specificity of 100%. The same was true for resistance to 3rd-generation cephalosporins in Enterobacteriaceae that constitutively produced class C β-lactamases. Discrimination was more difficult in species expressing class A β-lactamases, as these enzymes can generate false-positive results. Thus, the sensitivity and specificity for this group were 100% and 91.5%, respectively. The test permitted the prediction of resistance within 2.5 h after the blood culture was flagged as positive.     

Comparison of Three Kinds of Electrode–Skin Interfaces for Electrical Impedance Scanning

Low, uniform, and stable electrode–skin impedance is required to achieve good performance of the electrode–skin interface for electrical impedance scanning (EIS) examination. This can be used to measure the real impedance distribution of breast tissue beneath the skin. In this study, the gel interface, the cotton fine grid thin layer (CFGTL) interface, and the hydrogel interface were compared. Experiments were conducted to assess the influence of each interface on the multi-frequency EIS data and their capacity to retain moisture. Results showed that the CFGTL and hydrogel interfaces decreased contact impedance and made the impedance between the electrodes and the breast skin more even and stable. The Cole–Cole model was also used to fit the multi-frequency EIS data. The results demonstrated that the CFGTL and hydrogel interfaces were advantageous for measuring the impedance of the breast tissue under the gel interface. In general, the CFGTL and hydrogel interfaces had good contact with the skin, and both interfaces were proper choices for EIS examination at present. The hydrogel interface was a better choice for our new EIS system.     

Comparison and Evaluation of Three Animal Models for Studyingthe Pathogenicity of Staphylococcus epidermidis

Staphylococcus epidermidis was once thought as the normalflora of human skin that rarely causes disease in healthypersons, and isolation of this bacteria from clinical speci mens was generally taken as contamination. In recentyears, however, largely because of the increased use ofintra vascular catheters and other indwelling prosthetic de vices, S. epidermidis has emerged as a major nosocomialpathogen in biomaterial associated infections [1,2]. It hasbeen suggested that S. epidermid…     

Detection and characterization of Enterobacteriaceae producing metallo-β-lactamases in a tertiary-care hospital in Spain

Carbapenem-resistant or intermediate (MIC ≥1 mg/L) clinical isolates (n = 12) of three species of Enterobacteriaceae (Klebsiella pneumoniae, Klebsiella oxytoca and Escherichia coli) were characterized. The isolates harboured integrons containing the VIM-1 metallo-β-lactamase gene together with other resistance gene cassettes. In particular, the CTX-M-2 gene was detected in four of the K. pneumoniae isolates. The patient population was mostly paediatric and characterized by severe underlying illnesses that involved long-term hospitalization, major surgery and/or immunosuppressive and broad-spectrum antibiotic therapy.     

Point-Counterpoint: Can Newly Developed,Rapid Immunochromatographic Antigen Detection Tests Be Reliably Used for the Laboratory Diagnosis of Influenza Virus Infections?

Five years ago, the Point-Counterpoint series was launched. The initial article asked about the role of rapid immunochromatographic antigen testing in the diagnosis of influenza A virus 2009 H1N1 infection (D. F. Welch and C. C. Ginocchio, J Clin Microbiol 48:22–25, 2010, http://dx.doi.org/10.1128/JCM.02268-09). Since that article, not only have major changes been made in immunochromatographic antigen detection (IAD) testing for the influenza viruses, but there has also been rapid development of commercially available nucleic acid amplification tests (NAATs) for influenza virus detection. Further, a novel variant of influenza A, H7N9, has emerged in Asia, and H5N1 is also reemergent. In that initial article, the editor of this series, Peter Gilligan, identified two issues that required further consideration. One was how well IAD tests worked in clinical settings, especially in times of antigen drift and shift. The other was the role of future iterations of influenza NAATs and whether this testing would be available in a community hospital setting. James Dunn, who is Director of Medical Microbiology and Virology at Texas Children''s Hospital, has extensive experience using IAD tests for diagnosing influenza. He will discuss the application and value of these tests in influenza diagnosis. Christine Ginocchio, who recently retired as the Senior Medical Director, Division of Infectious Disease Diagnostics, North Shore-LIJ Health System, and now is Vice President for Global Microbiology Affairs at bioMérieux, Durham, NC, wrote the initial counterpoint in this series, where she advocated the use of NAATs for influenza diagnosis. She will update us on the commercially available NAAT systems and explain what their role should be in the diagnosis of influenza infection.     

Detection of IMP Metallo-β-Lactamase in Carbapenem-Nonsusceptible Enterobacteriaceae and Non-Glucose-Fermenting Gram-Negative Rods by Immunochromatography Assay

Metallo-β-lactamases (MBLs) are transmissible carbapenemases of increasing prevalence in Gram-negative bacteria among health care facilities worldwide. Control of the further spread of these carbapenem-resistant bacteria relies on clinical microbiological laboratories correctly identifying and classifying the MBLs. In this study, we evaluated a simple and rapid method for detecting IMP, the most prevalent MBL in Japan. We used an immunochromatography (IC) assay for 181 carbapenem-nonsusceptible (CNS) (nonsusceptible to imipenem or meropenem) strains comprising 74 IMP-producing and 33 non-IMP-producing strains of non-glucose-fermenting Gram-negative rods (NFGNR), as well as 64 IMP-producing and 10 non-IMP-producing Enterobacteriaceae strains. The IC assay results were compared to those from the double-disk synergy test (DDST), the MBL Etest, and the modified Hodge test (MHT) (only for Enterobacteriaceae). The IMP type was confirmed by specific PCR and direct sequencing. The IC assay detected all of the IMP-type MBLs, including IMP-1, -2, -6, -7, -10, -11, -19, -20, and -22 and IMP-40, -41, and -42 (new types), with 100% specificity and sensitivity against all strains tested. Although the sensitivity and specificity values for the DDST and MHT were equivalent to those for the IC assay, the MBL Etest was positive for only 87% of NFGNR and 31% of Enterobacteriaceae due to the low MIC of imipenem, causing an indeterminate evaluation. These results indicated that the IC assay might be a useful alternative to PCR for IMP MBL detection screening.     

Quantitative Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Rapid Resistance Detection

Antibiotic resistance in Gram-negative microorganisms is an increasing health care problem. The rapid detection of such resistance is crucial for starting an early specific therapy and to enable initiation of the required hygiene measures. With continued emphasis on reducing the cost of laboratory testing, only economical/low-cost approaches have a chance of being implemented. During recent years, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been developed to be a standard method in microbiology laboratories for the rapid and cost-efficient identification of microorganisms. Extending the usage of MALDI-TOF MS in the clinical microbiology laboratory to the area of resistance testing is an attractive option. Quantitative MALDI-TOF MS using an internal standard facilitates the measurement of the quantity of peptides and small proteins within a spectrum. These quantities correlate to the number of microorganisms and therefore to the growth of a microorganism. The comparison of growth in the presence or absence of an antibiotic allows for analysis of the susceptibility behavior of a strain. Here, we describe a novel method and its application in the analysis of 108 Klebsiella sp. isolates. After 1 h of incubation at a meropenem concentration of 8 μg/ml, a sensitivity of 97.3% and a specificity of 93.5% were achieved (compared to Etest results).     

Comparison of Lateral Flow Technology and Galactomannan and (1→3)-β-d-Glucan Assays for Detection of Invasive Pulmonary Aspergillosis

We compared a lateral flow device to galactomannan and (1→3)-β-d-glucan assays to detect invasive aspergillosis in an established guinea pig model of pulmonary disease. The lateral flow device became positive earlier (day 3) than the (1→3)-β-d-glucan and galactomannan assays (day 5), with all samples positive by each assay on day 7.Early diagnosis of invasive aspergillosis is critical for the initiation of appropriate antifungal therapy and may improve outcomes in high-risk patients (2). The use of sensitive biomarkers, including the noninvasive assays for galactomannan and (1→3)-β-d-glucan, also reduces the use of unnecessary antifungal agents (3, 5, 6). Despite their advantages, the galactomannan and the (1→3)-β-d-glucan assays are confined to laboratories equipped for these tests or require samples be sent to reference laboratories. Lateral flow technology incorporates immunochromatographic assays into simple devices for point-of-care diagnosis. When coupled to a monoclonal antibody specific to an extracellular glycoprotein of Aspergillus spp., this technology is a sensitive and specific biomarker (8). Our objective was to evaluate the time to positivity and sensitivity of a lateral flow device in an established guinea pig model of invasive pulmonary aspergillosis (9) and directly compare these results to those obtained using the galactomannan and (1→3)-β-d-glucan assays.Immunosuppressed male Hartley guinea pigs (Charles River Laboratories) were exposed to conidia for 1 h in an aerosol chamber (9). Serum samples were collected on days 3, 5, and 7 postinoculation. A previously described lateral flow device was used for the serodiagnosis of invasive aspergillosis (8). Briefly, an immunoglobulin G (IgG) monoclonal antibody (JF5) to an epitope on an extracellular antigen secreted constitutively during active growth of Aspergillus was immobilized to a capture zone on a porous nitrocellulose membrane. JF5 IgG was also conjugated to colloidal gold particles to serve as the detection reagent. Serum was added to a release pad containing the antibody-gold conjugate, which bound the target antigen, and then passed along the porous membrane and bound to JF5 IgG monoclonal antibody immobilized in the capture zone. Test results were available within 10 to 15 min after loading the sample. Bound antigen-antibody-gold complexes were observed as a red line with an intensity proportional to the antigen concentration and were classified as negative, weakly positive, moderately positive, or strongly positive (Fig. 1A, B, C, and D). Anti-mouse immunoglobulin immobilized to the membrane in a separate zone served as an internal control.Open in a separate windowFIG. 1.Examples of results from negative (A), weakly positive (B), moderately positive (C), and strongly positive (D) lateral flow device assays. In the absence of the Aspergillus antigen, no complex was formed in the zone containing solid-phase JF5 antibody, and a single internal control line was observed (A).The (1→3)-β-d-glucan assay was performed using a commercially available kit (Fungitell; Associates of Cape Cod) according to the manufacturer''s instructions. The mean rate of change in optical density (OD) at 405 nm over time was measured using a microplate spectrophotometer (Synergy HT; Biotek Instruments). Serum galactomannan was measured using a commercially available kit (Platelia Aspergillus enzyme immunoassay; Bio-Rad Laboratories) according to the manufacturer''s instructions. The OD values of each sample, positive control, negative control, and cutoff control were measured using a microplate spectrophotometer at 450 and 630 nm, and the galactomannan index was calculated as the OD of each sample divided by the mean cutoff of the control. The lateral flow assay and the (1→3)-β-d-glucan and galactomannan assays were performed in separate laboratories by different investigators blinded to the results of the other.For each biomarker, the time to positivity was defined as the first time point at which 20% of samples became positive. Time to positivity was plotted by Kaplan-Meier analysis, and differences in median time at which the assays became positive were analyzed by the log-rank test. Differences in the number of positive samples per time point between the assays were determined by Fisher''s exact test. The overall specificity of each assay was also measured in uninfected controls. All statistical tests were performed using Prism 5.0 (GraphPad Software, Inc.).The assays were negative 1 h postinoculation, prior to the onset of invasive disease, with the exception of a galactomannan test result (Table ​(Table1),1), which likely represented a false-positive result, as invasive disease was not yet established (9). Each biomarker became positive early, with more than three samples positive for each assay by day 5 postinoculation. In serial samples from the same animals, each biomarker continued to increase throughout the study (Fig. 2A, B, and C). When the weakly positive lateral flow device results were considered positive, the time to positivity for this assay occurred on day 3, which was significantly earlier than with the galactomannan (day 5; P = 0.03) and (1→3)-β-d-glucan (day 7; P < 0.001) assays. When the weakly positive lateral flow results were considered negative and only the moderately and strongly positive results as positive, the time to positivity for each biomarker assay occurred at the day 5 time point.Open in a separate windowFIG. 2.Results from serial serum samples collected over time from the same guinea pigs with invasive aspergillosis as measured by lateral flow technology (○), (1→3)-β-d-glucan assay (•), and galactomannan assay (▪). Each line represents the biomarker results from one animal at multiple time points. Serial samples were available for measurement of each biomarker at the multiple time points for six guinea pigs (GP 1 to 6). Symbols for the y axis of the lateral flow device graphs: +, weakly positive results; ++, moderately positive results; +++, strongly positive results.

TABLE 1.

Comparison of the lateral flow device and galactomannan and (1→3)-β-d-glucan assays