MALDI-TOF Mass Spectrometry for Multilocus Sequence Typing of <i>Escherichia coli</i> Reveals Diversity among Isolates Carrying <i>bla</i>_CMY-2-Like Genes

<div><p>Effective surveillance and management of pathogenic <i>Escherichia coli</i> relies on robust and reproducible typing methods such as multilocus sequence typing (MLST). Typing of <i>E</i>. <i>coli</i> by MLST enables tracking of pathogenic clones that are known to carry virulence factors or spread resistance, such as the globally-prevalent ST131 lineage. Standard MLST for <i>E</i>. <i>coli</i> requires sequencing of seven alleles, or a whole genome, and can take several days. Here, we have developed and validated a nucleic-acid-based MALDI-TOF mass spectrometry (MS) method for MLST as a rapid alternative to sequencing that requires minimal operator expertise. Identification of alleles was 99.6% concordant with sequencing. We employed MLST by MALDI-TOF MS to investigate diversity among 62 <i>E</i>. <i>coli</i> isolates from Sydney, Australia, carrying a <i>bla</i>_CMY-2-like gene on an IncI1 plasmid to determine whether any dominant clonal lineages are associated with the spread of this globally-disseminated resistance gene. Thirty-four known sequence types were identified, including lineages associated with human disease, animal and environmental sources. This suggests that the dissemination of <i>bla</i>_CMY-2-like-genes is more complex than the simple spread of successful pathogenic clones. <i>E</i>. <i>coli</i> MLST by MALDI-TOF MS, employed here for the first time, can be utilised as an automated tool for large-scale population analyses or for targeted screening for known high-risk clones in a diagnostic setting.</p></div

Bacterial strains used in this study.

Bacterial strains used in this study.</p

Examples of discrepancies between MLST by MALDI-TOF MS and sequencing.

<p>^a These alleles differ by the presence/absence of a 10,290 Da peak that is often not detected by the software but is visible upon manual inspection. Other alleles that differ by the presence/absence of this peak are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143446#pone.0143446.s005" target="_blank">S3 Table</a>.</p><p>^b Indistinguishable alleles have identical spectral patterns for all four cleavage reactions. Other indistinguishable alleles can be determined by the simulation software.</p><p>^c Workflow for discrepant alleles detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143446#pone.0143446.s001" target="_blank">S1 Fig</a>.</p><p>Examples of discrepancies between MLST by MALDI-TOF MS and sequencing.</p

Sequence types of <i>E</i>. <i>coli</i> strains carrying IncI1-<i>bla</i>_CMY-2 plasmids.

<p>^a Sequence type</p><p>^b Sequence type complex based on <a href="http://mlst.warwick.ac.uk/mlst/dbs/Ecoli" target="_blank">http://mlst.warwick.ac.uk/mlst/dbs/Ecoli</a>;–, no STC</p><p>^c One isolate has a new combination of alleles and one has a new <i>gyrB</i> variant.</p><p>Sequence types of <i>E</i>. <i>coli</i> strains carrying IncI1-<i>bla</i>_CMY-2 plasmids.</p

Addictive antibiotic resistance plasmids.

The replicon (rep, solid circle), antitoxin (AT, arrowhead) and toxin (T, arrow) genes of a PSK/addiction system, an antibiotic resistance gene (AbR) and corresponding antibiotic (Ab, solid blocks) are shown. (A) An addictive plasmid is stable in the absence of antibiotic selection. (B) An addictive plasmid can be displaced by an incompatible plasmid. (C) A compatible plasmid providing specific antitoxin (non-addictive compatible) leads to loss of addictive resistance plasmids from some cells. (D) An incompatible non-addictive interference plasmid providing specific antitoxin (non-addictive incompatible) ensures that all bacterial cells are ultimately free of both plasmid types.</p

<i>In vivo</i> cure of pEl1573-colonized mice.

<p><i>In vivo</i> cure of pEl1573-colonized mice.</p

Plasmid interference for curing antibiotic resistance plasmids <i>in vivo</i>

<div><p>Antibiotic resistance increases the likelihood of death from infection by common pathogens such as <i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria <i>in vitro</i> and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different <i>Enterobacteriaceae in vitro</i> and restored antibiotic susceptibility <i>in vivo</i> to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.</p></div

Exclusion and incompatibility.

<p>Replicon (solid circle), antitoxin and toxin genes (arrowhead, arrow) and antibiotic resistance genes (CTX^R, orange and TET^R, black solid blocks). Interference plasmid not excluded by entry exclusion system (EES) is incompatible (INC) with resident CTX^R plasmid and is selected by TET.</p

Protocol for mouse experiments.

<p>Protocol for mouse experiments.</p

<i>In vivo</i> cure of pEl1573-colonized mice.

<p><i>In vivo</i> cure of pEl1573-colonized mice.</p