Assessment of Phenotype Relevant Amino Acid Residues in TEM-ß-Lactamases by Mathematical Modelling and Experimental Approval.

Single substitutions or combinations of them alter the hydrolytic activity towards specific ß-lactam-antibiotics and ß-lactamase inhibitors of TEM-ß-lactamases. The sequences and phenotypic classification of allelic TEM variants, as provided by the NCBI National Database of Antibiotic Resistant Organisms, does not attribute phenotypes to all variants. Some entries are doubtful as the data assessment differs strongly between the studies or no data on the methodology are provided at all. This complicates mathematical and bioinformatic predictions of phenotypes that rely on the database. The present work aimed to prove the role of specific substitutions on the resistance phenotype of TEM variants in, to our knowledge, the most extensive mutagenesis study. In parallel, the predictive power of extrapolation algorithms was assessed. Most well-known substitutions with direct impact on the phenotype could be reproduced, both mathematically and experimentally. Most discrepancies were found for supportive substitutions, where some resulted in antagonistic effects in contrast to previously described synergism. The mathematical modelling proved to predict the strongest phenotype-relevant substitutions accurately but showed difficulties in identifying less prevalent but still phenotype transforming ones. In general, mutations increasing cephalosporin resistance resulted in increased sensitivity to ß-lactamase inhibitors and vice versa. Combining substitutions related to cephalosporin and ß-lactamase inhibitor resistance in almost all cases increased BLI susceptibility, indicating the rarity of the combined phenotype.

Fast, economic and simultaneous identification of clinically relevant Gram-negative species with multiplex real-time PCR.

AIM: A newly designed multiplex real-time PCR (rt-PCR) was validated to detect four clinically relevant Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa). MATERIALS & METHODS: Serial dilutions of genomic DNA were used to determine the limit of detection. Colony PCR was performed with isolates of the four selected species and other species as negative controls. Isolates were characterized genotypically and phenotypically to evaluate the assay. RESULTS: Specific signals of all target genes were detected with diluted templates comprising ten genomic equivalents. Using colony rt-PCR, all isolates of the target species were identified correctly. All negative control isolates were negative. CONCLUSION: The genes gad, basC, khe and ecfX can reliably identify these four species via multiplex colony rt-PCR.

ESBL- and AmpC-producing Escherichia coli from legally and illegally imported meat: Characterization of isolates brought into the EU from third countries.

Multiresistant Enterobacteriaceae producing extended-spectrum ß-lactamases (ESBL) constitute a serious healthcare concern. Contaminated meat and meat products have been suggested as a major transmission route for these strains in the population. In this study, 36 ESBL-/AmpC-producing Escherichia (E.) coli isolates recovered from meat products imported into the EU from third countries (non-EU countries), both legally and illegally, were examined with regard to their antibiotic resistance profiles, virulence-associated genes and their genetic relatedness. The isolates were characterized by antibiotic susceptibility testing, multilocus sequence typing, macrorestriction analysis, microarray analyses and additional PCR assays. The most prevalent ESBL gene among the isolates was blaCTX-M-2 (n = 15), followed by the AmpC ß-lactamase gene blaCMY-2 (n = 8). The remaining isolates carried genes belonging to CTX-M groups 8, 1 or 9, or blaSHV-12. This distribution differs from the genotypes typically detected in meat-associated isolates of European origin. Most isolates showed additional phenotypic resistances and genes conferring resistance to further antimicrobial agents were detected through microarray analysis. Most commonly observed were resistances to tetracycline, sulfamethoxazole/trimethoprim, and gentamicin. A genotype conferring multidrug resistance to 3 or more classes of antimicrobials could be observed in 33 isolates (91.7%). Most isolates carried at least one gene associated with virulence and one isolate could be identified as an enteropathogenic E. coli, indicating a potential risk to consumers' health. Molecular typing results revealed a genetic variety among the isolates. The most common multilocus sequence types were ST101 and ST117, represented by three isolates each. One isolate belonged to ST131 and three novel sequence types could be identified among three isolates (ST7509, ST7602, ST7845). Group D was the most prevalent phylogenetic group, which was represented by 18 isolates. Overall, the results of this study show that imported meat products can constitute a source for locally uncommon lineages of multidrug resistant and virulent ESBL-producing E. coli and can thereby facilitate their dissemination in Europe.

A multiplex real-time PCR for the direct, fast, economic and simultaneous detection of the carbapenemase genes blaKPC, blaNDM, blaVIM and blaOXA-48.

A novel multiplex real-time PCR was designed to detect the clinically most important carbapenemase genes, blaKPC, blaVIM, blaNDM and blaOXA-48. The multiplex assay was verified testing genomic DNA of 24 carbapenemase-producing strains. It was validated using a blinded panel of 82 carbapenemase-producing and 50 non-producing isolates by direct colony PCR.

Economic high-throughput-identification of influenza A subtypes from clinical specimens with a DNA-oligonucleotide microarray in an outbreak situation.

Influenza A surface proteins H (haemagglutinin) and N (neuraminidase) occur in sixteen and nine distinct genotypes, respectively. The need for a timely production of vaccinations in case of pandemics or seasonal epidemics requires rapid typing methods for the determination of these alleles. The aim of the present study was to develop and improve a rapid and economic assay for determining H and N subtypes of influenza A from patient samples. The assay is based on the hybridisation of labelled amplicons from H and N reverse transcriptase-PCRs using consensus primer pairs to subtype-specific probes on microtiterstripe-mounted DNA-microarrays. An algorithm for semi-automatic data interpretation of raw data and assignment to H and N subtypes was proposed. Altogether, 191 samples were genotyped. This included 134 patient and 44 reference samples as well as controls. Under routine conditions sensitivity and specificity proved to be comparable to conventional nested or real-time PCRs. At least 130 out of 147 array-positive samples were unambiguously assignable. This included all sixteen variants of H as well as all nine variants of N. Furthermore, eighty-two samples from the 2009/2010 "novel H1N1/swine flu" (SF)-outbreak were correctly identified.

Use of miniaturized protein arrays for Escherichia coli O serotyping.

Serological typing of Escherichia coli O antigens is a well-established method used for differentiation and identification of O serotypes commonly associated with disease. In this feasibility study, we have developed a novel somatic antibody-based miniaturized microarray chip, using 17 antisera, which can be used to detect bound whole-cell E. coli antigen with its corresponding immobilized antibody, to assess the feasibility of this approach. The chip was tested using the related 17 control strains, and the O types found by the microarray chip showed 100% correlation with the O types found by conventional typing. A blind trial was performed in which 100 E. coli isolates that had been O serotyped previously by the conventional assay were tested by the array approach. Overall, the O serotypes of 88% of isolates were correctly identified by the microarray method. For several isolates, ambiguity of O-type designation by microarray arose due to increased sensitivity of this method, allowing signal intensities of cross-reactions to be quantified. Investigation of discrepancies between conventional and microarray O serotyping indicated that some isolates upon storage had become untypeable and, therefore, gave poor signal intensity when tested by the microarray or retested by conventional means. For all 20 serotype O26 and O157 isolates, the apparent discrepancy in O serotyping was analyzed further by a third independent test, which confirmed the microarray results. Therefore, the use of miniaturized protein arrays increases the speed and efficiency of O serotyping in a cost-effective manner, and these preliminary findings suggest the microarray approach may have a higher accuracy than those of traditional O-serotyping methods.