Characterization of bacteriophages and their carriage in Staphylococcus aureus isolated from broilers in Poland.

1. The objective of this study was the isolation and morphological characterization of temperate bacteriophages induced from Staphylococcus aureus strains isolated from clinical samples from broiler chickens and turkeys. 2. Eighty-five S. aureus strains were tested for susceptibility to oxacillin in order to determine which were methicillin resistant (MRSA). A total of 24 strains showed resistance to methicillin. 3. Thirty-one bacteriophages that were lytic against S. aureus strains were isolated and the host range of the bacteriophages was evaluated. Based on the presence of a specific nucleotide sequence, molecular identification of bacteriophages was performed and the presence of genes responsible for the production of classical enterotoxins (A-E) was also analysed. 4. All the isolated bacteriophages had an icosahedral head and a long, thin, non-contractile flexible tail, characteristic of the family Siphoviridae of the order Caudovirales. Based on multiplex PCR results, the phages were found to belong to serogroups A, B and F (Fa, Fb subgroup), which include mostly temperate phages infecting S. aureus. 5. The titre of the phages ranged from 10-4 to 10-9 PFU/ml. The bacteriophages exhibited strong lytic properties against some of the strains of Staphylococcus. The broadest spectrum of activity against the strains was observed in the case of phages sa2, sa3, sa6, sa12, sa15 and sa21. 6. The PCR results showed that of the 31 bacteriophage DNA samples, 4 (12.9%) appeared to have enterotoxigenic genes.

A loop-mediated isothermal amplification procedure targeting the sodA gene for rapid and specific identification of Gallibacterium anatis.

This paper reports on the development and validation of a real-time loop-mediated isothermal amplification assay (LAMP) for rapid and specific identification of Gallibacterium anatis. To design a set of 6 primers using the LAMP technique, the conserved region of the G. anatis sodA gene was selected as a target. To evaluate primer specificity we used 120 field strains, the reference strain G. anatis ATCC 43329, and 9 non-G. anatis bacteria. The results confirmed positive reactions for all G. anatis strains tested by LAMP at 63°C for 60 min, with no cross-reactivity observed for the negative control bacteria, i.e., Haemophilus parainfluenzae (ATCC 51505 and ATCC 33392), Aggregatibacter aphrophilus ATCC 7901, Avibacterium endocarditis, Pasteurella multocida, Actinobacillus pleuropneumoniae, Avibacterium paragallinarum, Ornithobacterium rhinotracheale, and Escherichia coli. The lowest detectable amount of DNA for the LAMP reaction was 0.2561 pg, which was detected in about 34 min, while the highest available concentration of the G. anatis reference strain was detected in about 10 min. The lowest detectable amount of DNA for the real-time PCR reaction was 21.24 pg, which was detected in about 20 min, while the highest available concentration of the G. anatis reference strain was detected in about 7 min. Moreover, using the real-time LAMP assay the reaction could be effectively carried out in a volume of just 13 µL, about half the officially recommended reaction volume (25 µL). The aim of this study was to develop a highly sensitive and specific G. anatis real-time LAMP assay that is less time-consuming and less costly than quantitative PCR.

Factors responsible for subclinical mastitis in cows caused by Staphylococcus chromogenes and its susceptibility to antibiotics based on bap, fnbA, eno, mecA, tetK, and ermA genes.

The aim of this study was to recognize selected factors of virulence determining the adhesion of Staphylococcus chromogenes to cows' udder tissues in subclinical mastitis and to evaluate the susceptibility of this pathogen to antibiotics. The subjects of the study were 38 isolates of Staph. chromogenes from 335 samples of milk from cows with subclinical coagulase-negative staphylococci mastitis. Somatic cell count ranged between 216,000 and 568,000/mL of milk (average 356,000/mL of milk). We confirmed the ability to produce slime in 24 isolates (63.2%), and the ability to produce protease in 29 isolates (76.3%). In each slime-producing isolate, the bap gene was not found, and the fnbA and eno genes were not detected. In vitro tests showed that ceftiofur had the highest effectiveness against Staph. chromogenes (89.5% of susceptible isolates). Minimum inhibitory concentrations ranged from 0.06 to 2µg/mL for susceptible isolates. The minimum concentrations required to inhibit growth of 90 and 50% of the isolates for ceftiofur were at or below the cutoffs recommended by the Clinical and Laboratory Standards Institute (2 and 0.06µg/mL, respectively). A significant percentage of the isolates were susceptible to other ß-lactam antibiotics: amoxicillin with clavulanic acid (84.2%) and ampicillin (81.6%). The lowest effectiveness among ß-lactams was for penicillin (73.7% of susceptible isolates), and the minimum inhibitory concentration for penicillin ranged from <0.06 to 8µg/mL. None of the examined isolates had the mecA gene, but ß-lactamase was detected in 4 isolates (10.5%). Erythromycin and oxytetracycline exhibited the lowest activity against Staph. chromogenes (71.1 and 63.2% of susceptible isolates, respectively). The genes tetK (6 isolates) and ermA (1 isolate) were also detected.