Molecular Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates from Milk Samples of Dairy Cows with Mastitis in Bavaria, Germany.

The aim of this study was to determine the rate of extended-spectrum ß-lactamase (ESBL)-producing microorganisms among Escherichia coli isolates causing bovine mastitis, including molecular characterization of these isolates. Therefore, a total of 490 bovine E. coli isolates from milk samples of dairy cows with mastitis were investigated for ESBL production by antimicrobial susceptibility testing, PCR-based detection, and sequencing of ESBL encoding genes, which were identified in 22 isolates (4.5%). Moreover, resistance to the fluoroquinolones enrofloxacin and marbofloxacin occurred in 15 of 22 ESBL-producing isolates (68.2%). All ESBL-producing isolates carried a blaCTX-M-like gene, with blaCTX-M-14 (n = 10) as the most prevalent type. Seven isolates producing CTX-M-14 and belonging to phylogenetic group A were further investigated for genetic relatedness by multilocus sequence typing. Five of them could be assigned to four different sequence types (STs): ST10 (n = 2), ST167 (n = 1), ST410 (n = 1), and ST744 (n = 1), whereas the remaining two isolates could not be assigned. To conclude, the rate of ESBL-producing E. coli associated with cattle mastitis was 4.5%. Furthermore, a high proportion of fluoroquinolone coresistance could be detected. Therefore, careful and continuous surveillance of ESBL-producing E. coli in cattle and consequent implementation of prevention measures are needed to avoid a further spread of these multidrug-resistant bacteria.

MALDI-TOF MS based identification of food-borne yeast isolates.

In this study, food-borne yeast isolates (n=96), comprising at least 33 species, were identified using MALDI-TOF MS and conventional methods (API ID 32 C and Phoenix Yeast ID). Discrepancies of both methods were resolved by sequencing the ITS1-5.8S-rRNA-ITS2 region. For ten isolates, mainly classified to Rhodotorula and Trichosporon species, no clear final species identification was possible. 62 isolates were correctly identified to species level using either MALDI-TOF MS or conventional tests. 15 isolates were misidentified when applying conventional assays. In contrary, no species misidentifications were observed after MALDI-TOF MS based classification. In return, 16 isolates were not identifiable after matching their protein fingerprints against MALDI Biotyper 4.0.0.1 library. MALDI TOF MS in-house database update clearly improved the identification. In conclusion, the presented data suggest that MALDI-TOF MS is an appropriate platform for reliable classification and identification of food-borne yeast isolates.

PCR-SSCP-based reconstruction of the original fungal flora of heat-processed meat products.

Food processing of spoiled meat is prohibited by law, since it is a deception and does not comply with food safety aspects. In general, spoilage of meat is mostly caused by bacteria. However, a high contamination level of fungi could be also found in some meat or meat products with certain preserving conditions. In case that unhygienic meat is used to produce heat processed products, the microorganisms will be deactivated by heat, so that they cannot be detected by a standard cultivation method. Therefore, this study aimed to develop and apply a molecular biological method--polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP)--to reconstruct the original fungal flora of heat processed meat. Twenty primer pairs were tested for their specificity for fungal DNA. Since none of them fully complied with all study criteria (such as high specificity and sensitivity for fungal DNA; suitability of the products for PCR-SSCP) in the matrix "meat", we designed a new reverse primer, ITS5.8R. The primer pair ITS1/ITS5.8R amplified DNA from all tested fungal species, but not DNA from meat-producing animals or from ingredients of plant origin (spices). For the final test, 32 DNA bands in acrylamide gel from 15 meat products and 1 soy sauce were sequenced-all originating from fungal species, which were, in other studies, reported to contaminate meat e.g. Alternaria alternata, Aureobasidium pullulans, Candida rugosa, C. tropicalis, C. zeylanoides, Eurotium amstelodami and Pichia membranifaciens, and/or spices such as Botrytis aclada, Guignardia mangiferae, Itersonilia perplexans, Lasiodiplodia theobromae, Lewia infectoria, Neofusicoccum parvum and Pleospora herbarum. This confirms the suitability of PCR-SSCP to specifically detect fungal DNA in heat processed meat products, and thus provides an overview of fungal species contaminating raw material such as meat and spices.