日本の家畜と畜産農家から分離した mcr-1 によるコリスチン耐性大腸菌の分布状況と関連性について

Colistin is used to treat infectious diseases in humans and livestock; it has also been used as a feed additive for livestock for approximately 50 years. Since the mcr-1 plasmid-mediated colistin resistance gene was discovered in China in 2015, it has been detected worldwide, mainly in livestock. In this study, we investigated the prevalence and characteristics of mcr-mediated colistin-resistant Escherichia coli in livestock and farmers in Japan. We collected fecal samples from 295 healthy livestock (202 cattle and 93 swine) and 62 healthy farmers from 72 livestock farms (58 cattle farms and 14 swine farms) between 2013 and 2015. Twenty-eight mcr-1-harboring E. coli strains were isolated from 25 livestock (six cattle and 19 swine) and three farmers (two cattle farmers and one swine farmer). The prevalence rates of mcr-1-harboring E. coli in livestock and farmers were 8.47 and 4.84%, respectively. Of the 28 strains, the resistance genes of three were transferable via the mcr-1-coding plasmids to E. coli J53 at low frequencies (10-7-10-8). Six strains coharbored mcr-1 with CTX-M β-lactamases (CTX-M-14, CTX-M-27, or CTX-M-156). Of the isolates obtained from livestock and farmers in four farms (farms C, I, N, and P), nine strains had the same genotypical characteristics (sequence types and pulsed-field gel electrophoresis band patterns), plasmid characteristics (incompatibility group and plasmid transferability), and minimum inhibitory concentrations. Thus, the findings suggested that clonal strains could spread among livestock and farmers within farms. To our knowledge, this is the first study to detect clonal relatedness of mcr-1-mediated colistin-resistant E. coli in livestock and farmers. It is suggested that farmers are at a higher risk of acquiring mcr-1-harboring strains, calling for our attention based on the One Health concept.博士（医学）・甲第798号・令和3年9月29日Copyright © 2021 Nakano, Nakano, Nishisouzu, Suzuki, Horiuchi, Kikuchi-Ueda, Ubagai, Ono and Yano. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY https://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms

MICs (μg/mL) of antibiotics against <i>A</i>. <i>baumannii</i> ATCC 19606 and the clinical isolates of MDRA.

<p>MICs (μg/mL) of antibiotics against <i>A</i>. <i>baumannii</i> ATCC 19606 and the clinical isolates of MDRA.</p

Acinetobacter baumannii LOS Regulate the Expression of Inflammatory Cytokine Genes and Proteins in Human Mast Cells

Herein, we investigated the effect of bacterial lipooligosaccharides (LOS), from Acinetobacter baumannii, on the expression of pro-inflammatory genes that play an essential role in bacterial clearance. LAD2 human mast cells were stimulated with LOS derived from two strains of A. baumannii—ATCC 19606 and MDRA T14. LOS exposure induced the expression of genes for pro-inflammatory mediators, including TNF-α, IL-8, LTC4S, CCL4, and TLR4. The mRNA expression levels of a majority of the pro-inflammatory genes, except TLR4, in A. baumannii-LOS stimulated mast cells were increased. Moreover, co-culture of neutrophils with the supernatant obtained from LOS (ATCC 19606 and MDRA T14)-induced LAD2 cells increased the transmigration of neutrophils, which plays a critical role in the early protection against bacterial infections. The results of the present study suggest that LOS could be involved in the pathogenicity of A. baumannii by inducing inflammatory responses via mast cells and that IL-8 is involved in recruiting neutrophils in response to bacterial invasion

Effect of PMB at sub-MICs on the expression levels of efflux pumps and biofilm-related genes in <i>A</i>. <i>baumannii</i>.

<p>Summarized results showing the mRNA levels of efflux pumps and biofilm-related genes in strains ATCC 19606, R2 and R3 cultured in LB-broth with sub-MICs of PMB. The mRNA levels of (A) <i>adeB</i>, (B) <i>adeG</i>, (C) <i>adeJ</i>, (D) <i>ompA</i>, (E) <i>bap</i>, (F) <i>pgaA</i> and (G) <i>abaI</i> were analyzed by real-time PCR. Bar graph data are shown as the means ± SEM (n = 6) of 3 independent experiments. Asterisks indicate statistically significant differences (**<i>P</i>< 0.01; *<i>P<</i>0.05, non-treated bacteria <i>vs</i>. antibiotics-treated bacteria; One-way ANOVA).</p

Effect of CST at sub-MICs on the expression levels of efflux pumps and biofilm-related genes in <i>A</i>. <i>baumannii</i>.

<p>Summarized results showing the mRNA levels of efflux pumps and biofilm-related genes in strains ATCC 19606, R2 and R3 cultured in LB-broth with CST at sub-MICs. The mRNA levels of (A) <i>adeB</i>, (B) <i>adeG</i>, (C) <i>adeJ</i>, (D) <i>ompA</i>, (E) <i>bap</i>, (F) <i>pgaA</i> and (G) <i>abaI</i> were analyzed by real-time PCR. Bar graph data are shown as the means ± SEM (n = 6) of 3 independent experiments. Asterisks indicate statistically significant differences (**<i>P</i><0.01; *<i>P<</i>0.05, non-treated bacteria <i>vs</i>. antibiotics-treated bacteria; One-way ANOVA).</p

Relationship between biofilm formation and the expression of efflux pumps and biofilm-related genes in <i>A</i>. <i>baumannii</i> in the presence of PMB at its sub-MICs.

<p>Relationship between biofilm formation and the expression of efflux pumps and biofilm-related genes in <i>A</i>. <i>baumannii</i> in the presence of PMB at its sub-MICs.</p

Effect of four antibiotics at sub-MICs on bacterial growth and biofilm formation of <i>A</i>. <i>baumannii</i>.

<p>Summarized results showing the ratio of planktonic and biofilm cells in strain ATCC 19606 cultured in LB broth with (A) CST, (B) PMB, (C) MIN and (D) TGC at sub-MICs. Summarized results showing the ratio of planktonic and biofilm cells in strain R2 cultured in LB broth with (E) CST, (F) PMB, (G) MIN and (H) TGC at sub-MICs. Summarized results showing the ratio of planktonic and biofilm cells in strain R3 cultured in LB broth with (I) CST, (J) PMB, (K) MIN and (L) TGC at sub-MICs. Dark gray and gray bars indicate the ratio of planktonic and biofilm cells in <i>A</i>. <i>baumannii</i>, respectively. Bar graph data are shown as the mean ± SEM (n = 6) of 3 independent experiments. Asterisks indicate statistically significant differences in the number of planktonic cells (**<i>P</i>< 0.01; *<i>P</i>< 0.05, non-treated bacteria <i>vs</i>. antibiotics-treated bacteria; One-way ANOVA). Crosses indicate statistically significant differences in the number of biofilm cells (^††<i>P</i><0.01; ^†<i>P</i><0.05, non-treated bacteria <i>vs</i>. antibiotics-treated bacteria; One-way ANOVA).</p

Primers used for real-time PCR.

<p>Primers used for real-time PCR.</p