Background: The emergence and worldwide circulation of strains of beta-lactamase-producing Enterobacterales represent a major public health problem. The aim of this study was the characterization of extended spectrum beta-lactamase (ESBL) and carbapenemase-producing strains of Escherichia coli and Klebsiella pneumoniae resistant strains isolated in Kinshasa hospitals. Methods: Urine samples were collected from hospitalized and non-hospitalized patients. Bacteria were identified by using conventional biochemical tests and MALDI-TOF. Antibiotic susceptibility testing was performed by disk diffusion method. ESBL and carbapenemase phenotypes were screened using standard microbiological tests. ESBL-encoding genes were tested by multiplex end-point PCR assays and carbapenemase enzymes were identified by immunochromatography assay. Results: Out of 286 urines, 200 (69.9%) Gram negative bacteria were isolated, among them 70 (35.0 %) E. coli and 23 (11.5%) K. pneumoniae. ESBL were identified in 62.9 % (44/70) and in 52.2% (12/23) of E. coli and K. pneumoniae strains respectively. The rate of ESBL-producing Enterobacterales was 60.2% (56/93). Among ESBL producers, the blaCTX-M1 gene was identified in 88.6 % (39/44) isolates of E. coli and in 100% (12/12) isolates of K. pneumoniae. The bla-gene blaCTX-M-1 was identified in 91.2% (51/56) of all ESBL producers strains. Only 3.2% (3/93) of Enterobacterales strains produced the NDM enzyme. The Other genes encoding beta-lactamase were blaTEM (64.3 %), blaOXA-1/-30 (58.9 %), and blaSHV (39.3%). Conclusion: A high rate of ESBL-producing Enterobacterales was observed. The results of the study report the first description of metallo-β-lactamase NDM producing E. coli and K. pneumoniae strains from patients in Democratic Republic of Congo.

Abstract Background Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches. Methods 96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher). Results According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%). Conclusions The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.

Objectives: In order to elaborate a new national challenge panel of resistant Gram-negative bacilli and Gram-positive cocci strains for the validation of routine antimicrobial susceptibility testing (AST) methods, an interlaboratory evaluation was organised. Methods: The results of 12 well-characterised multidrug-resistant strains tested by nine laboratories using local disk diffusion (DD) and automated AST (AUST) methods were compared with the reference broth microdilution method. Results: Overall categorical agreement ranged from 70% to 100% both for DD and AUST and was >90% for all but one strain for all antibiotics. Conclusion: Our multicentre AST study showed good reproducibility and the panel can be used as national resistant reference strains for routine AST validation. (C) 2022 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.

OBJECTIVES:Following two studies conducted in 2005 and 2011, a third prevalence survey of multidrug-resistant microorganisms (MDRO) was organised in Belgian nursing homes (NHs) using a similar methodology. The aim was to measure the prevalence of carriage of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum β-lactamase producing Enterobacteriaceae (ESBLE) and carbapenemase-producing Enterobacteriaceae (CPE) in NH residents. Risk factors for MDRO carriage were also explored.METHODS:Up to 51 randomly selected residents per NH were screened for MDRO carriage by trained local nurses between June and October 2015. Rectal swabs were cultured for ESBLE, CPE and VRE, while pooled samples of nose, throat and perineum and chronic wound swabs were obtained for culture of MRSA. Antimicrobial susceptibility testing, molecular detection of resistance genes and strain genotyping were performed. Significant risk factors for MDRO colonization MDRO was determined by univariate and multivariable analysis.RESULTS:Overall, 1447 residents from 29 NHs were enrolled. The mean weighted prevalence of ESBLE and MRSA colonization was 11.3% and 9.0%, respectively. Co-colonization occurred in 1.8% of the residents. VRE and CPE carriage were identified in only one resident each. Impaired mobility and recent treatment with fluoroquinolones or with combinations of sulphonamides and trimethoprim were identified as risk factors for ESBLE carriage, while for MRSA these were previous MRSA carriage/infection, a stay in several different hospital wards during the past year, and a recent treatment with nitrofuran derivatives. Current antacid use was a predictor for both ESBL and MRSA carriage.CONCLUSIONS:In line with the evolution of MRSA and ESBL colonization/infection in hospitals, a decline in MRSA carriage and an increase in ESBLE prevalence was seen in Belgian NHs between 2005 and 2015. These results show that a systemic approach, including surveillance and enhancement of infection control and antimicrobial stewardship programs is needed in both acute and chronic care facilities.

Four screening assays aimed for rapid detection of carbapenemase production from Gram-negative bacterial isolates, i.e., the Neo-Rapid Carb kit (Rosco Diagnostica A/S), the Rapidec Carba NP test (bioMérieux SA), the β Carba test (Bio-Rad Laboratories N.V.), and a homemade electrochemical assay (BYG Carba test) were evaluated against a panel comprising 328 clinical isolates (Enterobacteriaceae [n = 198] and nonfermentative Gram-negative bacilli [n = 130]) with previously characterized resistance mechanisms to carbapenems. Among Enterobacteriaceae isolates, the BYG Carba test and the β Carba test showed excellent sensitivities (respectively, 100% and 97.3%) and specificities (respectively, 98.9% and 97.7%). The two other assays yielded poorer performances with sensitivity and specificity of 91.9% and 83.9% for the Rapidec Carba NP test and of 89.2% and 89.7% for the Neo-Rapid Carb kit, respectively. Among Pseudomonas spp., sensitivities and specificities ranged, respectively, from 87.3% to 92.7% and from 88.2% to 94.1%. Finally, all tests performed poorly against Acinetobacter spp., with sensitivities and specificities, respectively, ranging from 27.3% to 75.8% and from 75 to 100%. Among commercially available assays, the β Carba test appeared to be the most convenient for routine use and showed the best overall performances, especially against OXA-48-like producers. The excellent performance of the BYG Carba test against Enterobacteriaceae was confirmed (100% sensitivity and 98.9% specificity).

The study aimed to characterize beta-lactam resistance mechanisms of Enterobacteriaceae isolates recovered from diseased dogs and cats between 2008 and 2010 in a European surveillance program (ComPath I) for the antibiotic susceptibility of bacterial pathogens. A total of 608 non-duplicated Enterobacteriaceae isolates were obtained prior antibiotic treatment from diseased dogs (n=464) and cats (n=144). Among the 608 Enterobacteriaceae isolates, 22 presented a minimal inhibitory concentration against cefotaxime above EUCAST breakpoints of susceptibility. All the 22 isolates remained susceptible to carbapenems. Ten isolates were confirmed as extended-spectrum-beta-lactamase (ESBL) producers by PCR-sequencing of bla coding genes including 9 blaCTX-M (CTX-M-1, 14, 15, 32,…) and 1 blaTEM-52 and 12 were AmpC-producing isolates (10 plasmidic CMY-2 group and 2 isolates overexpressing their chromosomal AmpC). ESBLs and plasmid-mediated AmpC (pAmpC)-producing isolates were mainly recovered from dogs (n=17) suffering from urinary tract infections (n=13) and originated from eight different countries. ESBL-bearing plasmids were mostly associated with IncFII incompatibility groups while CMY-2 was predominantly associated with plasmid of the IncI1 group. ESBL/pAmpC-producing Escherichia coli belonged to phylogroup A (n=5), B2 (n=4), and D (n=5). Multilocus sequence typing analysis revealed that among three CTX-M-15-producing E. coli, two belong to sequence type (ST) 131 and one to ST405. The presence of CTX-M-15 including on IncFII plasmids in E. coli ST131-B2 has also been described in isolates of human origin. This suggests the possibility of exchanges of these isolates from humans to companion animals or vice-versa.

The ability of various combination disk tests (CDTs) incorporating avibactam to detect OXA-48 carbapenemase-producing Enterobacteriaceae was evaluated. The CDT using 30-μg temocillin alone and supplemented with 5-μg avibactam showed good performance and could be an adjunctive test to the classic CDT containing class A and class B carbapenemase inhibitors for the positive discrimination of OXA-48 carbapenemase producers from carbapenemase-negative strains.

OBJECTIVES:To assess the performance of the agar disc diffusion method for the detection of carbapenemase-producing Enterobacteriaceae (CPE) referred to the national reference laboratories (NRLs) in Belgium and France.METHODS:All Enterobacteriaceae isolates referred to the NRLs for the confirmation of CPE in 2012 were included. The inhibition zone diameters of meropenem, piperacillin/tazobactam and temocillin using CLSI disc diffusion methodology were recorded. Phenotypic and molecular detection of carbapenemases was performed on all isolates.RESULTS:A total of 1354 Enterobacteriaceae isolates, including 435 (32.1%) confirmed CPE isolates [OXA-48 (n = 323), KPC (n = 60), VIM (n = 32) and NDM (n = 20)] and 919 carbapenemase-negative isolates, were tested. Using recommended interpretative criteria, non-susceptibility to meropenem had poor sensitivity (52.0% by CLSI susceptibility breakpoint and 80.0% by EUCAST screening breakpoints), while non-susceptibility to piperacillin/tazobactam (according to CLSI breakpoint) or to temocillin (according to Fuchs, Barry, Thornsberry et al. Eur J Clin Microbiol 1985; 4: 30-3) was highly sensitive (99.8% and 98.2%, respectively) but poorly specific (29.4% and 42.9%, respectively) for the detection of CPE. Temocillin diameters <12 mm alone had high specificity (90.0%) and the combination of temocillin diameters ≥12 mm with piperacillin/tazobactam diameters ≥16 mm observed in 40% of all referred isolates displayed excellent negative predictive value (99.2%).CONCLUSIONS:In geographical areas with a high prevalence of OXA-48 producers, recommended meropenem susceptibility or screening breakpoints failed to detect CPE in a large proportion of isolates. The combination of modified zone diameter cut-offs for piperacillin/tazobactam (≥16 mm) and temocillin (≥12 mm) can be used to rule out the presence of carbapenemase and avoid unnecessary additional testing for confirmation of CPE.

ABSTRACT We compared the performance of the Carba NP test and the Rosco Rapid CARB screen kit for detecting carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa . Both tests are rapid and highly sensitive; however, the Carba NP test showed superior specificity, and several uninterpretable results were observed with the Rapid CARB screen.

OBJECTIVES:A national survey was conducted to determine the prevalence and risk factors of methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamases-producing Enterobacteriaceae (ESBLE) and vancomycin-resistant enterococci (VRE) among nursing home residents in Belgium.METHODS:A random stratified, national prevalence survey was conducted in nursing home residents who were screened for carriage of ESBLE, MRSA and VRE by multisite enriched culture. Characteristics of nursing homes and residents were collected by a questionnaire survey and were analysed by multilevel logistic regression analysis.RESULTS:Of 2791 screened residents in 60 participating nursing home, the weighted prevalence of ESBLE and MRSA carriage were 6.2% (range: 0 to 20%) and 12.2% (range: 0 to 36%), respectively. No cases of VRE were found. No relationship was found between ESBLE and MRSA prevalence rates within nursing homes and the rate of co-colonization was very low (0.8%). Geographical variations in prevalence of MRSA and ESBLE and in distribution of ESBL types in nursing home residents paralleled that of acute hospitals. Risk factors of ESBLE carriage included previously known ESBLE carriage, male gender, a low level of mobility and previous antibiotic exposure. Risk factors for MRSA colonization were: previously known MRSA carriage, skin lesions, a low functional status and antacid use.CONCLUSIONS:A low prevalence of ESBLE carriage was found in nursing home residents in Belgium. The prevalence of MRSA carriage decreased substantially in comparison to a similar survey conducted in 2005. A low functional status appeared as a common factor for ESBLE and MRSA carriage. Previous exposure to antibiotics was a strong predictor of ESBLE colonization while increased clustering of MRSA carriage suggested the importance of cross-transmission within nursing homes for this organism. These results emphasize the need for global coordination of the surveillance of MDRO within and between nursing homes and hospitals.

OBJECTIVES:To determine the point prevalence of carbapenem-non-susceptible Enterobacteriaceae (CNSE) and carbapenemase-producing Enterobacteriaceae (CPE) isolates among hospitalized patients in Belgium.METHODS:Twenty-four hospital-based laboratories prospectively collected 200 non-duplicated Enterobacteriaceae isolates from clinical specimens of hospitalized patients over a 2 month period. All isolates were screened locally for decreased susceptibility to carbapenem drugs using a disc diffusion method according to CLSI interpretative criteria. CNSE strains were referred centrally for confirmation of carbapenemase by phenotypic and molecular testing.RESULTS:From February to April 2012, 158 of the 4564 screened Enterobacteriaceae isolates were categorized as non-susceptible to carbapenems, resulting in a point prevalence of CNSE of 3.5% (95% CI: 2.9%-4.2%; range per centre: 0.5%-8.5%). Of the 125 referred CNSE isolates, 11 Klebsiella pneumoniae isolates [OXA-48 (n = 7), KPC type (n = 3) and NDM type (n = 1)], 1 OXA-48-positive Escherichia coli isolate and 1 KPC-positive Klebsiella oxytoca isolate were detected in eight hospitals. None of the 72 carbapenem-non-susceptible Enterobacter spp. isolates were confirmed as CPE. The minimal estimated point prevalence of CPE isolates was 0.28% (13/4564; 95% CI: 0.13%-0.44%) overall (range per centre: 0%-1.5%).CONCLUSIONS:Despite the overall low prevalence of CNSE found in this study, the detection of CPE isolates in one-third of the participating centres raises concerns and highly suggests the spread and establishment of CPE in Belgian hospitals.

OBJECTIVES:To assess the in vitro susceptibility of multidrug-resistant Enterobacteriaceae (MDRE) isolates to tigecycline.METHODS:Clinical isolates of MDRE tested in this study were obtained from 91 hospitals in Belgium during the period January 2010 to April 2010. MICs of tigecycline were determined by Vitek 2 (VTK) and by the reference broth microdilution (BMD) method, and the results were interpreted based on the 2011 MIC interpretative criteria recommended by EUCAST.RESULTS:A total of 501 non-duplicate MDRE isolates were tested. These comprised 284 isolates of Escherichia coli [255 (89.7%) were extended-spectrum β-lactamase (ESBL)-producing isolates], 72 isolates of Klebsiella pneumoniae [53 (73.6%) were ESBL-producing isolates], 72 isolates of Enterobacter aerogenes, 33 isolates of Enterobacter cloacae, 19 isolates of Klebsiella oxytoca and 21 miscellaneous others. The MIC(90) values of tigecycline for E. coli and non-E. coli ESBL-producing Enterobacteriaceae isolates were 0.5 and 2 mg/L by BMD, and 0.5 and 8 mg/L by VTK, respectively. The highest essential and categorical agreement rates between VTK and BMD results using EUCAST breakpoints were observed in E. coli isolates (97.2%), while lower and unacceptable essential and categorical agreement rates were obtained for isolates belonging to species other than E. coli (81.1% and 59.4%, respectively).CONCLUSIONS:VTK appears to be a suitable method for routine susceptibility testing of tigecycline only for E. coli isolates, while BMD should be preferred for other Enterobacteriaceae species isolates.

Objectives: To determine prevalence, incidence and risk factors of colonization by extended-spectrum beta-lactamase-producing Enterobacteriacae (ESBLE), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE) in aged subjects admitted to an acute geriatric unit at a teaching hospital.Methods: During 12 months, 337 patients were screened by nasal, oropharyngeal, groin, axillary and rectal swabs upon admission and at discharge.Results: The prevalence of ESBLE, MRSA and VRE carriage upon admission was 11.6%, 7.5% and 0.6%, respectively. The incidence density of ESBLE and MRSA carriage was respectively of 1.77 and 2.40 new cases for 1000 patient-days. No cases of VRE acquisition were found. Risk factors for ESBLE colonization on admission were: multiple contacts with the hospital within the previous year, chronic catheter use and a high level of dependency. For MRSA, risk factors were: chronic wounds, anti-acid use and a high level of dependency.Conclusion: This study shows a high prevalence of asymptomatic colonization of ESBL-producing Escherichia coli in patients admitted to an acute geriatric ward, as high as MRSA carriage. A low functional status is a common risk factor both for ESBLE and for MRSA colonization and it highlights the need to reinforce infection control measures. (C) 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

ABSTRACT Five multidrug-resistant nonclonally related Enterobacteriaceae isolates were recovered in Belgium in 2010 from three patients who had been hospitalized in Pakistan, Montenegro, and Serbia/Kosovo. New Delhi metallo-β-lactamase (NDM-1) was detected in each of the isolates in addition to several extended-spectrum β-lactamases (CTX-M-15, SHV-12), plasmidic cephalosporinases (CMY-16, CMY-58), rRNA methylases (ArmA, RmtB), and Qnr genes (qnrA6, qnrB1, qnrB2). One patient died from uncontrolled sepsis, while the two others recovered. No secondary cases occurred in any of the hospitals.

ABSTRACT The aim of this study was to evaluate the performance of Brilliance ESBL agar (OX; Oxoid, Basingstoke, United Kingdom), a novel chromogenic agar for the selective isolation and the presumptive identification of extended-spectrum-beta-lactamase (ESBL)-producing Enterobacteriaceae . A panel of 200 clinical Gram-negative Enterobacteriaceae and nonfermenting isolates with defined resistance mechanisms was inoculated onto OX and onto ChromID ESBL agar (BM; bioMérieux, Marcy l'Etoile, France) chromogenic medium in the first part of the study to evaluate the growth selectivity and chromogenic features of these two media. Of the 156 Enterobacteriaceae challenge isolates, 8 fully susceptible isolates were inhibited, all 98 ESBL producers were detected, and 50 isolates harboring other resistance mechanisms were recovered on both chromogenic agars. In the second phase, 528 clinical samples (including 344 fecal specimens) were plated onto OX, BM, and MacConkey agar with a ceftazidime disk (MCC) for the screening of ESBL-producing Enterobacteriaceae . Growth on at least one medium was observed with 144 (27%) of the clinical samples screened. A total of 182 isolates, including 109 (60%) of Enterobacteriaceae , were recovered and 70 of these (from 59 specimens) were confirmed as ESBL-producing isolates. The sensitivities of MCC, BM, and OX were 74.6%, 94.9%, and 94.9%, respectively. The specificities of MCC, BM, and OX by specimens reached 94.9%, 95.5%, and 95.7%, respectively, when only colored colonies were considered on the two selective chromogenic media. The high negative predictive value (99.3%) found for OX suggests that this medium may constitute an excellent screening tool for the rapid exclusion of patients not carrying ESBL producers.

BACKGROUND:Because of the increasing resistance of Helicobacter pylori against metronidazole and clarithromycin, alternative regimens including newer fluoroquinolones have been developed. We aimed to assess the prevalence as well as the mechanisms of this resistance in clinical isolates originating from patients living in Belgium. METHODS:Minimal inhibitory concentration (MIC) values of ciprofloxacin, levofloxacin, and moxifloxacin were determined by Etest method on 488 H. pylori isolates originating from patients who underwent upper gastrointestinal endoscopy at 10 different centers. Resistant strains (MIC values > 1 microg/ml) were evaluated for the presence of point mutations in the quinolone resistance-determining region (QRDR) of the gyrA by amplification and nucleotide sequence. RESULTS:Eighty-two (16.8%) of the strains were found resistant to all fluoroquinolones and 70 of these were further analyzed. Homogeneous and heterogeneous resistance were observed in 55 (78.6%) and in 15 (21.4%) of the strains, respectively. QRDR sequencing revealed various mutations of the codons corresponding to Asn-87 and Asp-91 in all isolates with homogeneous resistance. However, in 12 of 15 strains displaying heterogenous resistance, mutations were only detected after subcultures of isolated colonies growing within the ellipse inhibition zone of the E-test. Amino acid substitutions in the QRDR of GyrA could not be directly related with the MIC values of the isolates. Fluoroquinolone-resistant mutants were easily selected in vitro at frequencies ranging between 10(-6) and 10(-7). Such selected mutants stably persisted after several serial passage in antibiotic-free agar. CONCLUSIONS:These results suggest that H. pylori resistance to fluoroquinolones is occurring at a high frequency in the Belgian population and that it is essentially mediated through a variety of point mutations occurring in a few loci of GyrA. As a consequence, we strongly suggest to determine the susceptibility of the infecting isolates to fluoroquinolones before administration of an anti-H. pylori regimen including these agents.

ABSTRACT A novel chromogenic agar medium (ESBL-Bx; bioMérieux, Marcy l'Etoile, France) was compared to MacConkey agar supplemented with 2 mg ceftazidime/liter (MCKC) for the selective isolation and presumptive identification of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae directly from clinical samples. Of a total of 644 clinical specimens (including 551 fecal samples), 496 yielded no growth and 148 yielded growth on one or both media. Overall, 44 ESBL-producing Enterobacteriaceae strains ( Escherichia coli [ n = 17], Enterobacter aerogenes [ n = 17], Klebsiella spp. [ n = 5], and Citrobacter freundii [ n = 5]) were isolated from 37 specimens by a combination of both methods after 18 to 24 h of incubation. The sensitivities were 97.7 and 84.1% for ESBL-Bx and MCKC, respectively, with 43 ESBL-positive strains isolated as colored colonies from 36 specimens on ESBL-Bx versus 37 ESBL-positive organisms isolated from 32 specimens on MCKC. The specificities by specimens were 89 and 91% for ESBL-Bx and MCKC, respectively. On either one of the two media, natural AmpC-hyperproducing Enterobacter spp. ( n = 25) and Citrobacter spp. ( n = 14) were the most common false positives as well as non-ESBL-producing Klebsiella oxytoca ( n = 18) on ESBL-Bx and Morganella morganii ( n = 10) on MCKC. We conclude that ESBL-Bx is a sensitive and specific medium for the isolation of ESBL-producing Enterobacteriaceae from clinical samples. The main advantages of ESBL-Bx over MCKC reside in its chromogenic character and its sensitivity and selectivity, which enabled the recovery and presumptive identification of most ESBL-producing Enterobacteriaceae within 24 h and reduced by 27% the need for unnecessary identification and confirmation of ESBL testing when disregarding all colorless colonies growing on this medium.

OBJECTIVES:We evaluated the reliability of cefpirome/clavulanate (CD04) compared with ceftazidime/clavulanate (CD02) and cefotaxime/clavulanate (CD03) Oxoid combination discs for the detection of extended-spectrum beta-lactamases (ESBL) in several Enterobacteriaceae isolates, including Enterobacter spp.METHODS:Overall, a total of 105 ESBL-positive [positive double-disc synergy test (DDST)] and 94 ESBL-negative (negative DDST) Gram-negative isolates were evaluated. Ninety-eight isolates were confirmed as ESBL-positive on the basis of the sequence alignments of the blaTEM and/or blaSHV gene amplification products, which matched with previously identified ESBLs. The phenotypic detection of ESBLs was performed by the three combination discs according to the NCCLS and BSAC methods. The CD04 disc was evaluated with the manufacturer's recommended zone size difference breakpoint of > or =4 mm.RESULTS:In Escherichia coli and Klebsiella spp., the sensitivities (%)/specificities (%) of CD02, CD03 and CD04 discs, and the combination of CD02 or CD04 discs, were, respectively, 88/92, 90/92, 95/84 and 100/82, while the corresponding figures were 94/100, 4/100, 94/100 and 100/100 in Enterobacter aerogenes. NCCLS and BSAC methods yielded concordant results in 99% of the isolates.CONCLUSIONS:CD04 and CD02 discs were the best combination for detection of ESBLs in our collection of Enterobacteriaceae isolates, including E. aerogenes.