Antimicrobial resistance in isolates of non-human originMonday, April 02, 2012, 12:30 - 13:30
Isolation and characterisation of extended-spectrum beta-lactamase CTX-M bacteria from waste milk samples from dairy farms in England and Wales in 2011L. Randall*, M. Sharma, R. Horton, L. Brunton, T. Brightwell, C. Teale, J. Jones (New Haw, GB)
Objectives: The aim of this study was to analyse waste milk samples from farms in England and Wales for the presence of Extended Spectrum Beta-Lactamase (ESBL) bacteria.
Methods: Waste milk due for feeding to calves that day was collected from 103 farms and stored chilled for up to 24 hours whilst transported to the laboratory. Counts of bacteria in raw waste milk samples were made on blood agar (total aerobic count), CHROMagar ECC (total Enterobacteriaceae count), CHROMagar CTX (presumptive ESBLs) and CHROMagar ECC + 16 mg/L cefoxitin (mainly AmpC-producing bacteria). Milk samples were then incubated overnight in buffered peptone broth before plating on the above agars to detect presence / absence of bacteria. Presence of ESBL and AmpC phenotypes in isolates from selective agars was determined using appropriate MAST sensitivity disks. Isolates with an ESBL phenotype were tested for blaCTX-M, blaOXA, blaSHV and blaTEM genes, isolates with a blaCTX-M gene were sequenced to determine CTX-M sequence type and most ESBL phenotype presumptive E. coli isolates were serotyped. Bacteria were identified by MALDI-ToF Additionally, MICs of nine antibiotics were performed against representative ESBL isolates.
Results: Bacteria [presumptive E. coli] were isolated from 99%, 84.5% [43.7%], 62.14% [4.85%] and 20.39% [1.9%] of the 103 milk samples, on blood agar, CHROMagar ECC, CHROMagar ECC + 16 mg/L cefoxitin and CHROMagar CTX respectively before enrichment. Mean total [presumptive E. coli] counts per ml in raw milk were 1.42 x 107, 5.30 x 105 [1.94 x 104], 1.75 x 104 [3.22 x 102], 3.80 x 104 [5.10 x 102] on blood agar, CHROMagar ECC, CHROMagar ECC + 16 mg/L cefoxitin and CHROMagar CTX respectively. Maximum total aerobic counts per ml and total Enterobacteriaceae counts per ml were in the order of 109 and 107 respectively. A total of 6 milk samples was positive for CTX-M bacteria (Table 1). CTX-M sequence types 1, 14, 14b and 15 were present in isolates of Citrobacter, Enterobacter cloacae, E. coli and Kluyvera intermedia (Table 1). Some of the CTX-M isolates were highly resistant to cephalosporins (Table 1).
Conclusions: Waste milk samples from farms in England and Wales can have high bacterial counts. About 6% of waste milk samples in this study contained bacteria which carried the blaCTX-M gene. These results show that, if waste milk is fed to calves it may expose them to CTX-M bacteria.