Characterization of Multi-Drug Resistant <i>Enterococcus faecalis </i>Isolated from Cephalic Recording Chambers in Research Macaques (<i>Macaca</i> spp.)

<div><p>Nonhuman primates are commonly used for cognitive neuroscience research and often surgically implanted with cephalic recording chambers for electrophysiological recording. Aerobic bacterial cultures from 25 macaques identified 72 bacterial isolates, including 15 <i>Enterococcus faecalis</i> isolates. The <i>E</i>. <i>faecalis</i> isolates displayed multi-drug resistant phenotypes, with resistance to ciprofloxacin, enrofloxacin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, bacitracin, and erythromycin, as well as high-level aminoglycoside resistance. Multi-locus sequence typing showed that most belonged to two <i>E</i>. <i>faecalis</i> sequence types (ST): ST 4 and ST 55. The genomes of three representative isolates were sequenced to identify genes encoding antimicrobial resistances and other traits. Antimicrobial resistance genes identified included <i>aac(6’)-aph(2”)</i>, <i>aph(3’)-III</i>, <i>str</i>, <i>ant(6)-Ia</i>, <i>tetM</i>, <i>tetS</i>, <i>tetL</i>, <i>ermB</i>, <i>bcrABR</i>, <i>cat</i>, and <i>dfrG</i>, and polymorphisms in <i>parC</i> (S80I) and <i>gyrA</i> (S83I) were observed. These isolates also harbored virulence factors including the cytolysin toxin genes in ST 4 isolates, as well as multiple biofilm-associated genes (<i>esp</i>, <i>agg</i>, <i>ace</i>, <i>SrtA</i>, <i>gelE</i>, <i>ebpABC</i>), hyaluronidases (<i>hylA</i>, <i>hylB</i>), and other survival genes (<i>ElrA</i>, <i>tpx</i>). Crystal violet biofilm assays confirmed that ST 4 isolates produced more biofilm than ST 55 isolates. The abundance of antimicrobial resistance and virulence factor genes in the ST 4 isolates likely relates to the loss of CRISPR-<i>cas</i>. This macaque colony represents a unique model for studying <i>E</i>. <i>faecalis</i> infection associated with indwelling devices, and provides an opportunity to understand the basis of persistence of this pathogen in a healthcare setting.</p></div

Population Characteristics of Sampled Research Macaques.

<p>Population Characteristics of Sampled Research Macaques.</p

Biofilm Growth of <i>E</i>. <i>faecalis</i> in Flow Cell Chambers.

<p>Attached cells grown in flow cell chambers for 43 h were stained green with SYTO-9, and visualized at 63X magnification. Images are representative for each isolate.</p

24 hour Biofilm Production for 15 <i>E</i>. <i>faecalis</i> Isolates Assessed by Crystal Violet Staining.

<p>ST 4 and ST 330 isolates produce significantly more biofilm than ST 55 isolates (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169293#pone.0169293.s005" target="_blank">S4 Table</a>). Mean normalized OD₅₇₀ for pooled data were 2.016 ±.0.016 for ST 4 isolates, 1.500 ± 0.2942 for ST 55 isolates, 8.191 ± 0.1489 for the ST 330 isolate and 1.894 ± 0.1833 for the ATCC 29212 control strain.</p

Selected Antimicrobial Resistance MIC Results and Resistance Genes Identified from Whole Genome Sequence Data.

<p>Selected Antimicrobial Resistance MIC Results and Resistance Genes Identified from Whole Genome Sequence Data.</p

Aerobic Bacterial Culture Results from 45 Chambers¹of Research Macaques (n = 25).

<p>Aerobic Bacterial Culture Results from 45 Chambers<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169293#t003fn001" target="_blank">¹</a>of Research Macaques (n = 25).</p

The Majority of Macaque Chambers Display Polymicrobial Colonization.

<p>The Majority of Macaque Chambers Display Polymicrobial Colonization.</p

Kirby Bauer Disk Diffusion Results for 15 Macaque Chamber <i>E</i>. <i>faecalis</i> Isolates.

<p>Kirby Bauer Disk Diffusion Results for 15 Macaque Chamber <i>E</i>. <i>faecalis</i> Isolates.</p

Minimum Inhibitory Concentration (MIC) via Broth Microdilution and Etest Results for Macaque Chamber <i>E</i>. <i>faecalis</i> Isolates as Compared to ATCC <i>E</i>. <i>faecalis</i> 29212.

<p>Sequence types and historical recording chamber antimicrobial exposure are indicated for each macaque isolate.</p

Primers Used for the Identification and Characterization of <i>E</i>. <i>faecalis</i>.

<p>Primers Used for the Identification and Characterization of <i>E</i>. <i>faecalis</i>.</p