Carriage of Staphylococcus aureus by free-living wild animals in Spain

The presence of methicillin-susceptible Staphylococcus aureus (MSSA) was analyzed in different free-living wild animals to assess the genetic diversity and predominant genotypes on each animal species. Samples were taken from the skin and/or nares, and isolates were characterized by spa typing, multilocus sequence typing (MLST) and antimicrobial susceptibility testing. The proportion of MSSA carriers were 5.00, 22.93, 19.78, and 17.67% in Eurasian griffon vulture, Iberian ibex, red deer, and wild boar, respectively (P = 0.057). A higher proportion of isolates (P = 0.000) were recovered from nasal samples (78.51%) than skin samples (21.49%), but the 9.26% of red deer and 18.25% of wild boar would have been undetected if only nasal samples had been tested. Sixty-three different spa types were identified, including 25 new spa types. The most common were t528 (43.59%) in Iberian ibex, t548 and t11212 (15.79% and 14.04%) in red deer, and t3750 (36.11%) in wild boar. By MLST, 27 STs were detected, of which 12 had not been described previously. The most frequent were ST581 for Iberian ibex (48.72%), ST425 for red deer (29.82%), and ST2328 for wild boar (42.36%). Isolates from Eurasian griffon vulture belong to ST133. Host specificity has been observed for the most frequent spa types and STs (P = 0.000). The highest resistance percentage was found against benzylpenicillin (average, 22.2%), although most of the S. aureus isolates were susceptible to all antimicrobial tested. Basically, MSSA isolates were different from those MRSA isolates previously detected in the same animal species.

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.