3.6. Measuring susceptibility/resistance to antibiotics of Paenibacillus larvae

In some countries, the antibiotic oxytetracycline (OTC) has been used by beekeepers for decades to prevent and control AFB in honey bee colonies as an alternative to the burning of infected beehives in areas where disease incidence is high. However, the intensive use of tetracyclines in professional beekeeping resulted in tetracycline-resistant (TcR) and oxytetracycline-resistant (OTCR) P. larvae isolates. There is now general concern about widespread resistance involving horizontal-transfer via non-genomic (i.e. plasmid or conjugal transposon) routes and also induced resistance by the presence of sub-inhibitory concentrations of tetracycline (Alippi et al., 2007). P. larvae highly resistant phenotypes have been correlated with the presence of natural plasmids carrying different Tc resistance determinants, including tetK and tetL genes (Murray and Aronstein, 2006; Alippi et al., 2007; Murray et al., 2007).

Most Paenibacillus species, including P. larvae, are highly susceptible to tetracyclines; it has been reported that the growth of P. larvae strains is inhibited at concentrations as low as 0.012 µg of oxytetracycline per ml of culture medium. Alternatively, when a disc containing 5 µg of oxytetracycline is placed on an agar plate previously spread with a bacterial suspension, the clear zones formed by the sensitive strains usually average 50 mm in diameter including the disc (Shimanuki and Knox, 2000). Any reduction of the inhibition zone or an increase in the minimal inhibitory concentration (MIC) required to prevent the growth of P. larvae would be evidence of the development of resistant strains.

3.6.2. Determination of antibiotic susceptibility testing by the disc diffusion method