Application of a luminescent bacterial biosensor for the detection of tetracyclines in routine analysis of poultry muscle samples

Tetracyclines are extensively used in veterinary medicine. For the detection of tetracycline residues in animal products, a broad array of methods is available. Luminescent bacterial biosensors represent an attractive inexpensive, simple and fast method for screening large numbers of samples. A previously developed cell-biosensor method was subjected to an evaluation study using over 300 routine poultry samples and the results were compared with a microbial inhibition test. The cell-biosensor assay yielded many more suspect samples, 10.2% versus 2% with the inhibition test, which all could be confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Only one sample contained a concentration above the maximum residue limit (MRL) of 100 mu g kg-1, while residue levels in most of the suspect samples were very low (10 mu g kg-1). The method appeared to be specific and robust. Using an experimental set-up comprising the analysis of a series of three sample dilutions allowed an appropriate cut-off for confirmatory analysis, limiting the number of samples and requiring further analysis to a minimu

Ionophore resistance and potential risk of ionophore driven co-selection of clinically relevant antimicrobial resistance in poultry

Today’s intensive broiler production is highly dependent on ionophore coccidiostats. These antiparasitic agents are applied as feed additives. The most important group, the ionophores, not only exhibit antiparasitic activity but are also antimicrobial against Gram-positive bacteria. Because these ionophores are not used in humans, it is widely assumed their use does not impact human health. However, recent Swedish and Norwegian research shows that ionophores can cause the co-selection of vancomycin resistance in enterococci. Vancomycin is a critically important antimicrobial for human medicine. Because of its limited relevance for human medicine, monitoring of antimicrobial resistance against ionophores, in particular salinomycin (SAL), was discontinued several years ago. From historical data, however, it can be retrieved that a fair percentage of the Enterococcus faecium and Enterococcus faecalis isolates from poultry origin show SAL resistance. The current research was initiated to determine whether ionophore resistance in enterococci from poultry co-occurs with other clinically relevant types of antimicrobial resistance. A set of 137 E. faecium and E. faecalis isolates from poultry products from conventional and organic origin was phenotypically characterized. The resistance profiles were statistically analysed and subsequently a selection of 20 isolates was DNA sequenced and analysed for the presence of resistance genes. Almost 40% of the isolates appeared to have a SAL resistant phenotype. Results of the statistical analysis showed a significant correlation between the presence of SAL resistance and erythromycin (ERY) resistance in E. faecium from conventional farming. The same correlation was also observed in E. faecalis from both conventional and organic origin. Besides this, both E. faecium and E. faecalis show significant correlation between the presence of SAL and tetracycline (TET) resistance and SAL and ampicillin (AMP) resistance. DNA sequencing results show good agreement between the observed phenotypic resistance and the identified resistance genes. In all of the isolates showing phenotypic SAL resistance the narAB genes could be identified. Moreover, in most isolates narAB is found to be located on a plasmid carrying additional genes for other types of antibiotic resistance. This is an alarming observation, since it implies that the use of ionophores may drive the transfer and dissemination of other, clinically relevant types of antimicrobial resistance by co-selection. These results question the sustainability of the prophylactic use of ionophores in broiler production. More research is needed to determine the extent and the impact of this issue. It is unknown whether ionophore resistance also occurs in other Gram-positive bacteria, like Staphylococcus aureus. It is plausible that ionophore resistance genes other than narAB will be present in poultry associated bacteria. E. faecium and E. faecalis are an important cause of hospital-acquired infections. Analysis of human enterococcal isolates should reveal whether transmission from the poultry reservoir is occurring and thus whether the use of ionophores in poultry impacts human healthcare

Screening methods for the detection of antibiotic residues in slaughter animals: comparison of ther european union Four-Plate Test, the Nouws Antibiotic Test and the Premi Test (applied to muscle and kidney

Microbial growth inhibition tests are widely used as the primary screening approach for the detection of antibiotic residues in slaughter animals. In this study we evaluated and compared the performance of the European Union Four-Plate Test (EU4pt), the Nouws Antibiotic Test (NAT), and a commercial ampoule test, the Premi(R)Test (applied to both muscle and kidney), by parallel analysis of 735 slaughter animals. The EU4pt only showed significant inhibition with two muscle samples containing 305 mu g kg(-1) doxycycline and 648 mu g kg(-1) tulathromycin, while an maximum residue limit (MRL) violation of 1100 mu g kg(-1) sulfamethazine remained unnoticed. Premi(R)Test-muscle only detected the sulfamethazine containing sample, all other (1.1%) suspect samples appeared false-positive results. The same test applied to kidney yielded 4.1% suspect samples, while the NAT screening (based on analysis of renal pelvis fluid) showed 4.9% suspect results. The vast majority of these samples contained tetracycline and/or aminoglycoside residues. Premi(R)Test-kidney appeared to be more sensitive to aminoglycosides than the NAT screening, which failed to detect an MRL violation of 870 mu g kg(-1) gentamicin in kidney. Detection of less than MRL levels of tetracycline residues by the NAT proved its suitability for this residue group. Whether Premi(R)Test is sufficiently sensitive for accurate tetracycline detection in kidney remains doubtful, although changing over to kidney definitely improved the suitability of Premi(R)Test for the detection of residues in slaughter animals