Real-time PCR versus MALDI-TOF MS and culture-based techniques for diagnosis of bloodstream and pyogenic infections in humans and animals.

AIMS: This study was applied to evaluate the usefulness of a high-throughput sample preparation protocol prior to the application of quantitative real-time PCR (qPCR) for the early diagnosis of bloodstream and pyogenic infections in humans and animals compared to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and classical culture. METHODS AND RESULTS: Saponin-mediated selective host cell lysis combined with DNase-1 was applied for processing of whole blood and pus clinical samples collected from suspected cases of septicaemia and pyogenic infections in humans and animals. The pre-PCR processing strategy enabled the recovery of microbial cells with no changes in their colony forming units immediately after the addition of saponin. DNase-1 was efficient for removing the DNAs from the host cells as well as dead cells with damaged cell membranes. The metagenomic qPCR and MALDI-TOF MS could identify the bacterial community of sepsis at species level with a concordance of 97·37% unlike the conventional culture. According to qPCR results, Staphylococcus aureus (24·24%) was predominated in animal pyogenic infections, whereas Klebsiella pneumonia (31·81%) was commonly detected in neonatal sepsis. CONCLUSIONS: Saponin combined with DNase-1 allowed the efficient recovery of microbial DNA from blood and pus samples in sepsis using qPCR assay. SIGNIFICANCE AND IMPACT OF THE STUDY: Metagenomic qPCR could identify a broad range of bacteria directly from blood and pus with more sensitivity, higher discriminatory power and shorter turnaround time than those using MALDI-TOF MS and conventional culture. This might allow a timely administration of a prompt treatment.

Coexistence of plasmid-mediated quinolone resistance determinants and AmpC-Beta-Lactamases in Escherichia coli strains in Egypt.

Three kinds of plasmid­mediated quinolone resistance (PMQR) determinants (qnr genes, qepA and aac(6')­Ib­cr) have been discovered and shown to be widely distributed among clinical isolates. To characterize the prevalence of PMQR determinants among AmpC­producing E. coli strains in food­producing animals and animal by­products in Egypt, twenty­nine E. coli strains were tested for their susceptibilities to antimicrobials and screened for PMQR determinants and AmpC Beta lactamases using PCR and plasmid profiling. It was found that qnr genes being detected alone or in combination with qepA or aac(6')­Ib­cr genes in 11 (37.9%) strains comprising 9 for qnrA and only one for both qnrB and qnrS. Moreover, qepA and aac(6')­Ib­cr were detected in 41.38% and 3.45% of E. coli strains, respectively. The ampC ß­lactamase genes were detected in 75.86 % of all strains and in 100% and 53.3% of the PMQR determinant­positive and negative strains, respectively. In several cases, plasmid profiling of E. coli strains exhibiting the coexistence of both PMQR determinants and ampC genes on a single plasmid as a first report in Egypt that may contribute to rapid spread and increase in bacterial resistance, which is important to public health concern.