PML) for the detection and confirmation of strains producing extended spectrum β-lactamases (ESBL)

Wissenschaftliches Programm 55. DGHM-Tagung 29. September-l. Oktober 2003 in Dresden Abstracts- Poster

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aim of this study was the validation of a PCR assay to detect Mycoplasrna spp. in primary sterile body fluids of horses and dogs with special consideration of methods to prevent false negative results due to inhibitors. A previously described PCR assay for Mycoplasma spp. and Acholeplasma spp. detection in cell cultures (Tang et al. 2000) was adapted for routine use. Samples were collected from horses (33 liquor, 58 synovia, 26 EDTAblood) and dogs (23 liquor, 28 synovia, 24 EDTAblood). DNA of liquor and synovia was released by boiling. DNA from EDTA-blood was prepared with QIAamp blood-Kit R. Six different Mycoplasma spp. and one Acholeplasrna sp. were used to evaluate the PCR assay. Using an internal positive control, only 7 of 192 samples (1 liquor, 4 synovia, 2 EDTA-blood) were found to inhibit the PCR reaction. The limit of detection of M. canis in samples from dogs and M. equigenitalium in samples from horses did not differ from control reactions with M. canis or M. equigenitalium in phosphate buffered saline. We modified and evaluated a robust nested PCR for the detection of Mycoplasma spp. in different body fluids of horses and dogs. With a specially designed internal control, false negative reactions due to inhibitors present in samples can be readily recognized.

Validation of the new Etest ESBL strip containing cefepime ± clavulanic acid ( P M / P M L ) for the detection and confirmation of strains producing extended spectrum ~-Iactamases (ESBL) St0renburg, E.1; Noor, D.1; Sobottka, 1.1; Laufs, R}, Mack, D. 2

i Universit~tsklinikurn Hamburg-Eppendorf; Institut for Infektionsmedizin 2Universit~tsklinikum Hamburg-Eppendorf; Institut f#r Medizinische Mikrobiologie und Immunologie I ntrod uction: In this study we evaluated the performance of a new Etest ESBL strip based on cefepime 4- clavulanic acid (PM/PML) in comparison to genotype characterisation. Methods: Etest PM/PML (AB Biodisk, Solna, Sweden) was compared to established Etest strips containing ceftazidime 4- clavulanic acid (TZ/TZL) and cefotaxime 4- clavulanic acid (CT/CTL). 41 clinical strains (10 E. coil, 25 K. pneumoniae, 3 K. oxytoca, and 3 E. aerogenes) ESBL positive by NCCLS criteria were used. PCR and sequencing have confirmed that all strains carry an ESBL enzyme (30 SHV, 7 CTX-M, and 4 TEM). 9 additional strains (3 E. coil, 6 K. oxytoca) with ESBL suspect antibiogram phenotypes but without PCRdetectable ESBL were also included in this evaluation. Data: Differences in ESBL substrate preferences were observed. Etest PM/PML gave the best ESBL detection

(98% sensitivity) with only one ESBL organism being missed. By using CT/CTL (TZ/TZL) strips 4 (11) ESBL strains were missed (90 and 73% sensitivity,

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Wissenschaftliches Programm 55. DGHM-Tagung 29. September-1. Oktober 2003 in Dresden Abstracts - Poster

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respectively). When ESBL interpretation was based on results of two strips simultaneously (e.g., TZ/TZL and CT/CTL, as recommeded by the manufacturer), the following results were obtained: CT/CTL + TZ/TZL 90% sensitivity, CT/CTL + PM/PML 98% sensitivity, TZ/TZL + PM/PML 98% sensitivity. PM/PML was the only strip capable of detecting ESBL activity in the three E. aerogenes strains. It is important to observe that ESBL results involve reading the MIC values to calculate the ratio of MIC reduction by clavulanic acid, as well as observing phantom (P) zones and deformation (D) of ellipses which are directly indicative of ESBL production. This was particularly important when using PM/PML. Conclusions. Etest PM/PML strip appeared to be a very sensitive test for ESBL detection, even as a stand-alone test. It was easy to use, but reading requires experience with respect to P and D effects. We particularly recommed the use of PM/PML for detection of ESBL in Enterobacter spp.

Establishment of a Capture-ELISA to detect the Beta2-toxin of CIostridium perfringens Sommer, A.1; Schoepe, H.1; Baljer, G. 1

~Justus-Liebig-Universitaet Giessen; Institut fuer Hygiene und Infektionskrankheiten der Tiere The first isolation of a novel Clostridium perfringens (C. p.)-toxin, the Beta2 ([32)-toxin occured in 1997. Till now only a [32-PCR to amplify the toxin-gene is described. The purpose of the present study was to establish a phenotypic testing system using 62-toxinspecific monoclonal antibodies (mAb), which is able to detect [32-toxin in supernatants of cultures of C. p. field isolates, directly. After immunization of Balb/c mice with a recombinant /32-toxin (r[32), two stable monoclonal hybridomas could be established which produced specific mAbs (4A5, 4 C l l ; IgG1) against the r[32 as well as against the native 132. The binding site of both mAbs is localized on different epitopes of the [32-toxin (Epitope-ELISA). The mAbs 4A5 and 4 C l l are used as capture antibodies in a [32-toxin specific Capture-ELISA, separately. A polyclonal [32-specific rabbit serum served as second antibody. The optimum binding capacity of the mAbs on NuncMaxi-Sorp-plates(R) was 0,3 IJg/100 tJI for mAb 4A5 and 0,4 tJg/100 IJI for mAb 4 C l l , respectively. The anti]32 rabbit serum was used in a 1:500 dilution in PBS, pH 8.0. For control r[32 and supernatants of a 132toxin positive C. p. type A-strain (18/00) were used in every test approach. Supernatants of 18 C. p. field isolates, all of wich were tested positive in the [32-specific PCR, specific 132reactivity with Capture-ELISA and immunoblot was found. Using the mAb 4A5 the [32-toxin detection limit can be stated at 0,0025 IJI/ml, regarding the mAb 4 C l l the limit is observed at 0,01 IJI/ml. No crossreactivities with other CIostridium species (C. bifermentans, C. septicum, C. p. type A and type C), grampositive (Staph. aureus, Bacillus cereus, Listeria monocytogenes, Sc. dysgalactiae ssp. equisimilis) and http://www.dghm.org

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