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.

Small-plasmid-mediated antibiotic resistance is enhanced by increases in plasmid copy number and bacterial fitness

Plasmids play a key role in the horizontal spread of antibiotic resistance determinants among bacterial pathogens. When an antibiotic resistance plasmid arrives in a new bacterial host, it produces a fitness cost, causing a competitive disadvantage for the plasmid-bearing bacterium in the absence of antibiotics. On the other hand, in the presence of antibiotics, the plasmid promotes the survival of the clone. The adaptations experienced by plasmid and bacterium in the presence of antibiotics during the first generations of coexistence will be crucial for the progress of the infection and the maintenance of plasmid-mediated resistance once the treatment is over. Here we developed a model system using the human pathogen Haemophilus influenzae carrying the small plasmid pB1000 conferring resistance to β-lactam antibiotics to investigate host and plasmid adaptations in the course of a simulated ampicillin therapy. Our results proved that plasmid-bearing clones compensated for the fitness disadvantage during the first 100 generations of plasmid-host adaptation. In addition, ampicillin treatment was associated with an increase in pB1000 copy number. The augmentation in both bacterial fitness and plasmid copy number gave rise to H. influenzae populations with higher ampicillin resistance levels. In conclusion, we show here that the modulations in bacterial fitness and plasmid copy number help a plasmid-bearing bacterium to adapt during antibiotic therapy, promoting both the survival of the host and the spread of the plasmid.

Characterization of plasmids in a human clinical strain of Lactococcus garvieae

The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25) encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.