Comparative molecular profiling of antimicrobial resistance and phylogenetic characterization of multidrug-resistant Escherichia coli isolated from meat sources in 2009 and 2021 in Japan

Predominance of Multidrug-Resistant Gram-Negative Bacteria Isolated from Supermarket Retail Seafood in Japan

“…Bacterial isolation was performed as previously described [13]. Briefly, a 25 g portion of each sample was weighed into a sterile stomacher bag and homogenized in 225 mL buffered peptone water (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 2% NaCl.…”

“…Total genomic DNA was extracted from the boiled lysates. Molecular screening of the 412 isolates was performed to identify extended-spectrum β-lactamase (ESBL)-encoding genes (bla SHV and bla CTX-M ), other β-lactamases (bla TEM , bla OXA-1 , bla OXA-2 , bla OXA-5 , bla OXA-9 , and AmpC), integrons (classes 1, 2, and 3), and aminoglycoside acetyltransferase gene aac(6')-Ib, as previously described [13,17]. PCR detection of mobile colistin-resistance genes (mcr-1-10) and acquired carbapenemases (KPC, NDM, OXA-48, VIM, and IMP) was performed [13].…”

“…Molecular screening of the 412 isolates was performed to identify extended-spectrum β-lactamase (ESBL)-encoding genes (bla SHV and bla CTX-M ), other β-lactamases (bla TEM , bla OXA-1 , bla OXA-2 , bla OXA-5 , bla OXA-9 , and AmpC), integrons (classes 1, 2, and 3), and aminoglycoside acetyltransferase gene aac(6')-Ib, as previously described [13,17]. PCR detection of mobile colistin-resistance genes (mcr-1-10) and acquired carbapenemases (KPC, NDM, OXA-48, VIM, and IMP) was performed [13]. The variable regions of classes 1 and 2 integrons within the integron positive isolates were amplified using primers complementary to the 5 and 3 conserved segments of class 1 integrons (5 CS and 3 CS) and class 2 integrons (hep74 and hep51).…”

“…The variable regions of classes 1 and 2 integrons within the integron positive isolates were amplified using primers complementary to the 5 and 3 conserved segments of class 1 integrons (5 CS and 3 CS) and class 2 integrons (hep74 and hep51). PCR amplification conditions were as follows: initial denaturation at 98 • C for 1 min, 30 cycles of denaturation at 98 • C for 10 s, annealing at 50 • C for 15 s, extension at 68 • C for 2 min, and final extension at 68 • C for 15 s [13]. PCR products were purified using ExoSAP-IT (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) and processed for Sanger sequencing using Eurofins Genomics (Tokyo, Japan).…”

“…Antimicrobial resistance (AMR) among seafood-borne bacteria can potentially decrease the effectiveness of antimicrobial therapy in humans and lead to human death, financial burden, and a major reduction in aquaculture production. Estimates suggest that approximately 10 million lives and USD 100 trillion will be lost by 2050 because of AMR if existing control measures to prevent the spread of AMR are compromised [13,14].…”