First Evidence of the Presence of the Causative Agent of Caseous Lymphadenitis-Corynebacterium pseudotuberculosis in Dairy Products Produced from the Milk of Small Ruminants.

This study focused on the detection and quantification of selected bacteria and on the presence of enterotoxin genes in milk and dairy products from sheep and goat farms in the Czech Republic using quantitative real-time PCR (qPCR) and multiplex PCR (PCR). The presence of Corynebacterium pseudotuberculosis (CP), Mycobacterium avium subsp. paratuberculosis (MAP), Listeria monocytogenes, Staphylococcus aureus, S. aureus enterotoxin genes and methicillin-resistant Staphylococcus aureus (MRSA) was determined in 18 milk samples, 28 fresh cheeses, 20 ripened cheeses and 14 yoghurts. The serological status of the herds in relation to CP and MAP was taken into account. The most frequently detected bacterium was S. aureus (48.8%), and subsequent PCR revealed 11 MRSA positive samples. The S. aureus enterotoxin genes seg, sei and sec were detected in two goat cheeses. Cheese samples showed a statistically higher risk of SA and MRSA occurrence. CP (8.8%) and MAP (13.8%) were detected by qPCR on two different seropositive farms. Cultivation of qPCR positive CP samples on agar plates supplemented with potassium tellurite showed the presence of viable bacterium. The results obtained confirmed the necessity of monitoring the infectious status of dairy animals and rapid diagnosis of bacterial pathogens in milk and dairy products.

Identification of and discrimination between the Mycobacterium abscessus complex and Mycobacterium avium complex directly from sputum using quadruplex real-time PCR.

Introduction. Cystic fibrosis (CF) is a serious disease with multisystemic clinical signs that is easily and frequently complicated by bacterial infection. Recently, the prevalence of nontuberculous mycobacteria as secondary contaminants of CF has increased, with the Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) being the most frequently identified. The MABSC includes subspecies of significant clinical importance, mainly due to their resistance to antibiotics.Gap statement. Sensitive method for early detection and differentiation of MABSC members and MAC complex for use in routine clinical laboratories is lacking. A method based on direct DNA isolation from sputum, using standard equipment in clinical laboratories and allowing uncovering of possible sample inhibition (false negative results) would be required. The availability of such a method would allow accurate and accelerated time detection of MABSC members and their timely and targeted treatment.Aim. To develop a real time multiplex assay for rapid and sensitive identification and discrimination of MABSC members and MAC complex.Methodology. The method of DNA isolation directly from the sputum of patients followed by quadruplex real-time quantitative PCR (qPCR) detection was developed and optimised. The sensitivity and limit of detection (LOD) of the qPCR was determined using human sputum samples artificially spiked with a known amount of M. abscessus subsp. massiliense (MAM).Results. The method can distinguish between MAC and MABSC members and, at the same time, to differentiate between M. abscessus subsp. abscessus/subsp. bolletii (MAAb/MAB) and MAM. The system was verified using 61 culture isolates and sputum samples from CF and non-CF patients showing 29.5 % MAAb/MAB, 14.7 % MAM and 26.2 % MAC. The LOD was determined to be 1 490 MAM cells in the sputum sample with the efficiency of DNA isolation being 95.4 %. Verification of the qPCR results with sequencing showed 100 % homology.Conclusions. The developed quadruplex qPCR assay, which is preceded by DNA extraction directly from patients' sputum without the need for culturing, significantly improves and speeds up the entire process of diagnosing CF patients and is therefore particularly suitable for use in routine laboratories.