Adaptive Evolution of Escherichia coli to an α-Peptide/β-Peptoid Peptidomimetic Induces Stable Resistance.

Antimicrobial peptides (AMPs) and synthetic analogues thereof target conserved structures of bacterial cell envelopes and hence, development of resistance has been considered an unlikely event. However, recently bacterial resistance to AMPs has been observed, and the aim of the present study was to determine whether bacterial resistance may also evolve against synthetic AMP analogues, e.g. α-peptide/β-peptoid peptidomimetics. E. coli ATCC 25922 was exposed to increasing concentrations of a peptidomimetic (10 lineages), polymyxin B (10 lineages), or MilliQ water (4 lineages) in a re-inoculation culturing setup covering approx. 500 generations. All 10 lineages exposed to the peptidomimetic adapted to 32 × MIC while this occurred for 8 out of 10 of the polymyxin B-exposed lineages. All lineages exposed to 32 × MIC of either the peptidomimetic or polymyxin B had a significantly increased MIC (16-32 ×) to the selection agent. Five transfers (≈ 35 generations) in unsupplemented media did not abolish resistance indicating that resistance was heritable. Single isolates from peptidomimetic-exposed lineage populations displayed MICs against the peptidomimetic from wild-type MIC to 32 × MIC revealing heterogeneous populations. Resistant isolates showed no cross-resistance against a panel of membrane-active AMPs. These isolates were highly susceptible to blood plasma antibacterial activity and were killed when plasma concentrations exceeded ≈ 30%. Notably, MIC of the peptidomimetic against resistant isolates returned to wild-type level upon addition of 25% plasma. Whole-genome sequencing of twenty isolates from four resistant lineages revealed mutations, in murein transglycosylase D (mltD) and outer-membrane proteins, which were conserved within and between lineages. However, no common resistance-conferring mutation was identified. We hypothesise that alterations in cell envelope structure result in peptidomimetic resistance, and that this may occur via several distinct mechanisms. Interestingly, this type of resistance result in a concomitant high susceptibility towards plasma, and therefore the present study does not infer additional concern for peptidomimetics as future therapeutics

Poor Invasion of Trophoblastic Cells but Normal Plaque Formation in Fibroblastic Cells despite actA Deletion in a Group of Listeria monocytogenes Strains Persisting in Some Food Processing Environments▿

We determined mammalian cell invasion and virulence gene (inlA, inlB, and actA) sequences of Listeria monocytogenes strains belonging to a molecular subtype (RAPD 9) that often persists in Danish fish-processing plants. These strains invaded human placental trophoblasts less efficiently than other L. monocytogenes strains, including clinical strains, and they carry a premature stop codon in inlA. Eight of 15 strains, including the RAPD 9 and maternofetal strains, had a 105-nucleotide deletion in actA that did not affect cell-to-cell spread in mouse fibroblasts. The RAPD 9 strains may still be regarded as of low virulence with respect to human listeriosis

Genome Sequencing Identifies Two Nearly Unchanged Strains of Persistent Listeria monocytogenes Isolated at Two Different Fish Processing Plants Sampled 6 Years Apart

Listeria monocytogenes is a food-borne human-pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities. Here we report the genome sequences for two L. monocytogenes strains (N53-1 and La111) that were isolated 6 years apart from two different Danish fish processers. Both strains are of serotype 1/2a and belong to a highly persistent DNA subtype (random amplified polymorphic DNA [RAPD] type 9). We demonstrate using in silico analyses that both strains belong to the multilocus sequence typing (MLST) type ST121 that has been isolated as a persistent subtype in several European countries. The purpose of this study was to use genome analyses to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical, with a predicted protein homology of 99.94%, differing at only 2 proteins. No single-nucleotide polymorphism (SNP) differences were seen between the two strains; in contrast, N53-1 and La111 differed from the EGD-e reference strain by 3,942 and 3,471 SNPs, respectively. We included a persistent L. monocytogenes strain from the United States (F6854) in our comparisons. Compared to nonpersistent strains, all three persistent strains were distinguished by two genome deletions: one, of 2,472 bp, typically contains the gene for inlF, and the other, of 3,017 bp, includes three genes potentially related to bacteriocin production and transport (lmo2774, lmo2775, and the 3′-terminal part of lmo2776). Further studies of highly persistent strains are required to determine if the absence of these genes promotes persistence. While the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicates that subtypes with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype

Minimum Inhibitory Concentration (µg/mL) of the adapted <i>E. coli</i> lineages against peptidomimetic 1 immediately after peptidomimetic adaptation and after 35 generations with no peptidomimetic.

a<p>MIC values are based on two technical duplicates.</p>b<p>Not determined due to lack of growth of revived freezing stocks in supplemented media.</p>c<p><i>E. coli</i> ATCC 25922 had a wt MIC of 8 µg/mL.</p

Heterogeneity in lineage populations.

<p>Minimum Inhibitory Concentration (MIC) for peptidomimetic 1 against population isolates of lineage no. 2 (A), lineage no. 4 (B), lineage no. 5 (C) and lineage no. 7 (D). Bars indicate biological replicates; MIC for all isolates was determined twice, a third replicate was performed for isolates 2–3 and 2–8 due to large variations in results. Susceptibility to peptidomimetic 1 varies widely within the populations. Solid line: population MIC; punctuated line: wt MIC (8 µg/mL).</p

Distribution of single-nucleotide polymorphisms (SNPs) and deletion-insertion polymorphism (DIPs) that caused an amino acid change and had a frequency above 80% in the peptidomimetic-adapted genome-sequenced isolates.

a1<p>isolates with high levels of resistance i.e. 8–16×wild type MIC, ² isolates with intermediate levels of resistance i.e. 4–8×wt MIC; ³ Isolates with wild type MIC.</p>b<p>Two SNPs were present in the <i>macB</i> gene: one SNP causing amino acid 319 in the protein to change from Asp to Tyr (X) and one causing amino acid 505 to change from Trp to Leu (x).</p>c<p>Two SNPs were present in the <i>macA</i> gene: one SNPs causing amino acid 91 in the protein to change from Val to Gly (X) and one causing amino acid 205 to change from Val to Leu (x).</p>d<p>DIP mutations; all other mutations are SNPs.</p