Table_2_Spread of the mcr-1 colistin-resistance gene in Escherichia coli through plasmid transmission and chromosomal transposition in French goats.XLSX

IntroductionColistin-resistance widely disseminated in food-producing animals due to decades of colistin use to treat diarrhea. The plasmid-borne mcr-1 gene has been extensively reported from bovine, swine and chicken worldwide, but smaller productions such as the goat farming sector were much less surveyed.MethodsWe looked for colistin-resistant isolates presenting plasmid-borne genes of the mcr family in both breeding (n=80) and fattening farms (n=5). Localization of the mcr-1 gene was performed using Southern blot analysis coupled to short-read and long-read sequencing.ResultsOnly the mcr-1 gene was identified in 10% (8/80) of the breeding farms and four over the five fattening farms. In total, 4.2% (65/1561) of the animals tested in breeding farms and 60.0% (84/140) of those tested in fattening farms presented a mcr-1-positive E. coli. The mcr-1 gene was located either on the chromosome (32.2%) or on IncX4 (38.9%) and IncHI2 (26.8%) plasmids. As expected, both clonal expansion and plasmidic transfers were observed in farms where the mcr-1 gene was carried by plasmids. Tn6330 transposition was observed in the chromosome of diverse E. coli sequence types within the same farm.DiscussionOur results show that the mcr-1 gene is circulating in goat production and is located either on plasmids or on the chromosome. Evidence of Tn6330 transposition highlighted the fact that chromosomal insertion does not impair the transmission capability of the mcr-1 gene. Only strict hygiene and biosecurity procedures in breeding farms, as well as a prudent use of antibiotics in fattening farms, can avoid such complex contamination pathways.</p

Table_1_Spread of the mcr-1 colistin-resistance gene in Escherichia coli through plasmid transmission and chromosomal transposition in French goats.XLSX

IntroductionColistin-resistance widely disseminated in food-producing animals due to decades of colistin use to treat diarrhea. The plasmid-borne mcr-1 gene has been extensively reported from bovine, swine and chicken worldwide, but smaller productions such as the goat farming sector were much less surveyed.MethodsWe looked for colistin-resistant isolates presenting plasmid-borne genes of the mcr family in both breeding (n=80) and fattening farms (n=5). Localization of the mcr-1 gene was performed using Southern blot analysis coupled to short-read and long-read sequencing.ResultsOnly the mcr-1 gene was identified in 10% (8/80) of the breeding farms and four over the five fattening farms. In total, 4.2% (65/1561) of the animals tested in breeding farms and 60.0% (84/140) of those tested in fattening farms presented a mcr-1-positive E. coli. The mcr-1 gene was located either on the chromosome (32.2%) or on IncX4 (38.9%) and IncHI2 (26.8%) plasmids. As expected, both clonal expansion and plasmidic transfers were observed in farms where the mcr-1 gene was carried by plasmids. Tn6330 transposition was observed in the chromosome of diverse E. coli sequence types within the same farm.DiscussionOur results show that the mcr-1 gene is circulating in goat production and is located either on plasmids or on the chromosome. Evidence of Tn6330 transposition highlighted the fact that chromosomal insertion does not impair the transmission capability of the mcr-1 gene. Only strict hygiene and biosecurity procedures in breeding farms, as well as a prudent use of antibiotics in fattening farms, can avoid such complex contamination pathways.</p

Schematic diagram of the ICE_<i>515_tRNA^Lys</i> mobile genetic element.

<p>ORFs appear as arrows. Genes encoding the putative bacteriocin system appear in light blue. The genes encoding the proteins that could be involved in oxidative stress response (NRAMP protein and thioredoxin-like) are colored in yellow and the gene encoding the putative new hemolytic CAMP factor (CAMP factor II) in purple. Genes encoding the proteins with LPxTG motif appear in pink. Genes of the conjugation module are indicated with blue arrows and regulation module with green arrows. Putative <i>oriT</i> is indicated by a star. Genes encoding a putative toxin-antitoxin system appear in orange and other genes encoding proteins with unknown function in white. The gene where the element is integrated (tRNA^Lys) is indicated in red. Recombination module is colored in red. Recombination sites are drawn as vertical rectangles. Black rectangles indicate identical sequences found in <i>attL</i>, <i>attR</i>, and <i>attI</i> sites; yellow rectangles indicate the arm of <i>attR</i> sites and the related arm of <i>attI</i> sites; and red rectangles indicate the arm of <i>attL sites</i> and the related arms of <i>attI</i> sites.</p

Expression of CAMP factor II in pathogenic streptococcal strains.

<p>A. CAMP test using (1) <i>S. bovis</i> 1052 wt, (2) <i>S. bovis</i> (pOri23-camp⁵¹⁵), (3) <i>S. dysgalactiae</i> 593 wt, (4) <i>S. dysgalactiae</i> (pOri23-camp⁵¹⁵) and (5) GBS strain 515 as control; B. co-hemolytic activity was monitored in <i>S. bovis</i> 1052 (filled circle with pOri23-camp⁵¹⁵ and empty circle without) and in <i>S. dysgalactiae</i> (filled squares with pOri23-camp⁵¹⁵ and empty squares without treatment). Hemolytic activity was measured at OD₆₃₀ every 30 min using a microplate reader. The experiment was done in triplicate using three independent biological samples. Errors bars represent the standard deviation observed between the 9 values obtained for each strain. Controls without SMase treatment were carried out (data not shown).</p

Comparison of CAMP factors and Uberis factor found in streptococci.

<p>A. multiple sequence alignment (<i>Sag</i>, <i>S. agalactiae</i>; <i>S. urinalis</i>; <i>S. canis</i>; <i>S. pyogenes</i>; <i>S. porcinus</i>; <i>S. pseudo porcinus</i> and <i>S. uberis</i>); B. phylogenetic tree showing the evolutionary relationships between the sequences of the alignment. Sequence alignment and construction of the phylogenetic tree were done using the AlignX module of VectorNTI advance 11 (InVitrogen). Conserved residues appear in light grey and identical amino acids appear in dark grey in the alignment. Position of residues in the sequence is indicated above the sequence. Sequence identities go from 56% (CAMP factor II of <i>S. agalactiae</i> and Uberis factor of <i>S. uberis</i>) to 100% (CAMP factor II of <i>S. agalactiae</i> and CAMP factor of <i>S. urinalis</i>). The CAMP factor of <i>P. acnes</i> is more distant (less than 30% of identity) and thus does not appear in this alignment. The phylogenetic tree has been constructed using the Neighbor Joining Method. Each branch of the tree has a length equal to the number of substitutions required to get from one nod to the next.</p

Analysis of the expression of the CAMP factor II (SAL_2074) gene.

<p>RT-PCR were performed using, as templates, RNA extracted from stationary-phase cultures of strain 515 (1, and negative control without RTase, 4); strain NEM316 (2 and negative control without RTase, 5); and transconjugant NEM316 (ICE_<i>515_tRNA^Lys</i>) (3 and negative control without RTase, 6). The same results were obtained in exponential phase. A positive control using genomic DNA of strain 515 was included (7) as well as a negative control with water (8). DNA molecular weight marker is marker number VI (Roche Applied Science).</p

Co-hemolytic activity of CAMP factor II expressed in <i>L. lactis</i> MG1363.

<p>A. CAMP test using (1) <i>L. lactis</i> MG1363 (pOri23), (2) <i>L. lactis</i> MG1363 (pOri23-camp⁵¹⁵) and (3) GBS strain 515 as control; B. <i>L. lactis</i> co-hemolytic activity was monitored in <i>L. lactis</i> MG1363 (pOri23) (filled circle with SMase treatment and empty circle without treatment) and in <i>L. lactis</i> MG1363 (pOri23-camp⁵¹⁵) (filled squares with SMase treatment and empty squares without treatment). Hemolytic activity was measured at OD₆₃₀ every 30 min using a microplate reader. The experiment was done in triplicate using three independent biological samples. Errors bars represent the standard deviation observed between the 9 values obtained for each strain. Controls without SMase treatment were done (data not shown).</p