When considering foodborne pathogens, bacteria that produce extended spectrum β-lactamases (ESBL) may pose a significant public health risk since the prevalent CTX-M type is cephalosporin resistant.1 Currently, there is no standard approach for the rapid, accurate detection of ESBL-producing bacteria in meat. Researchers have relied upon culture-based techniques, including cefpodoxime discs1, agars treated with cefotaxime and/or augmentin,2,3,4 and commercially available selective agars. Unfortunately, these approaches require long incubation and assay times and may even render insensitive results.
In this work, Anjum et al.5 develop a loop-mediated isothermal amplification (LAMP) assay capable of detecting extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (CTX-M groups 1, 2, and 9 and OXA-10-like genes) derived from meat samples.
The researchers found that it took between 6 and 38 minutes to produce positive results, based on strain, and that the LAMP assays achieved 100% specificity for the target isolates. The detection limit for CTX-M group 1, the most sensitive assay, was 41 gene copies per microliters. The detection limits for CTX-M group 2, CTX-M group 9, and OXA-10-like were 109, 169, and 74 gene copies per microliters, respectively. When the LAMP assays were applied to overnight bacterial cultures, the detection limits for all assays were between 104 and 105 cfu/mL crude lysate.
For chicken meat samples, the LAMP assays were faster and more sensitive with crude DNA extract instead of fresh BPW. The assays demonstrated 100% sensitivity and specificity for samples without competitor bacteria present (CTX-M groups 2 and 9) and correctly identified ESBL type. For CTX-M group 1 genes, LAMP assay detected targets in 7 out of 9 chicken samples. For OXA-10-like genes, LAMP assay detected targets in 0 out of 3 chicken samples at 10 cfu/g and 1 out of 3 chicken samples at 100 cfu/mL when competitors were present. In the non-chicken meat samples, the LAMP assays demonstrated 100% sensitivity and specificity for ESBLs in BPW with competitor bacteria present.
The researchers also reported that all agar types demonstrated 100% sensitivity and specificity for ESBLs after enrichment. They observe that both commercially prepared chromogenic agars, CHROMagar and Brilliance ESBL (Thermo Scientific) outperformed standard agar with antibiotics.
The cefpodoxime disc method demonstrated 100% specificity but only 86.7% sensitivity. The method failed to detect ESBL producers in 4 chicken samples (10 cfu/g). In addition to being less sensitive than the other methods, this approach required more time output than direct plating.
Overall, Anjum et al. found that the LAMP assay presented distinct advantages. The option to use overnight enrichment broth for the detection of ESBLs provides results a full 24 hours earlier than other methods. The LAMP assay also allows researchers to identify ESBLs by type without follow up assays. They note that the LAMP assays were sometimes less sensitive in the presence of competitor bacteria and recommend a combination of LAMP assay and culture-based techniques, preferably with chromogenic agar, for vigorous screening of meat products.
Learn more about Thermo Scientific Brilliance ESBL agar here
References
1 Warren, R.E. et al. (2008) ‘Imported chicken meat as a potential source of quinolone resistant Escherichia coli producing extended-spectrum beta-lactamases in the UK.’ Journal of Antimicrobial Chemotherapy 61(3):504–8.
2 Machado, E. et al. (2008) ‘Antibiotic resistance integrons and extended-spectrum {beta}-lactamases among Enterobacteriaceae isolates recovered from chickens and swine in Portugal.’ Journal of Antimicrobial Chemotherapy 62(2):296–302.
3 Mesa, R.J. et al. (2006) ‘Extended-spectrum beta-lactamase-producing Enterobacteriaceae in different environments (humans, food, animal farms and sewage).’ Journal of Antimicrobial Chemotherapy 58(1):211–5.
4 Dhanji, H. et al. (2005) ‘Cephalosporin resistance mechanisms in Escherichia coli isolated from raw chicken imported into the UK.’ Journal of Antimicrobial Chemotherapy 65(12):2534–7.
5 Anjum, M.F. et al. (2013) ‘Isolation and Detection of Extended Spectrum β-Lactamase (ESBL)-Producing Enterobacteriaceae from Meat using Chromogenic Agars and Isothermal Loop-Mediated Amplification (LAMP) Assays.’ Journal of Food Science 78 (12): M1892-1898.
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