Beta-Lactam Sensitive Bacteria Can Acquire ESBL-Resistance via Conjugation after Long-Term Exposure to Lethal Antibiotic Concentration

Abstract

Beta-lactams are commonly used antibiotics that prevent cell-wall biosynthesis. Beta-lactam sensitive bacteria can acquire conjugative resistance elements and hence become resistant even after being exposed to lethal (above minimum inhibitory) antibiotic concentrations. Here we show that neither the length of antibiotic exposure (1 to 16 h) nor the beta-lactam type (penam or cephem) have a major impact on the rescue of sensitive bacteria. We demonstrate that an evolutionary rescue can occur between different clinically relevant bacterial species (Klebsiella pneumoniae and Escherichia coli) by plasmids that are commonly associated with extended-spectrum beta-lactamase (ESBL) positive hospital isolates. As such, it is possible that this resistance dynamic may play a role in failing antibiotic therapies in those cases where resistant bacteria may readily migrate into the proximity of sensitive pathogens. Furthermore, we engineered a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-plasmid to encode a guiding CRISPR-RNA against the migrating ESBL-plasmid. By introducing this plasmid into the sensitive bacterium, the frequency of the evolutionarily rescued bacteria decreased by several orders of magnitude. As such, engineering pathogens during antibiotic treatment may provide ways to prevent ESBL-plasmid dispersal and hence resistance evolution.

Plasmid Viability Depends on the Ecological Setting of Hosts within a Multiplasmid Community

“…While the plasmid cost appears to have only a minor effect on the growth rate or host density in the media used in the experiment, it is still possible that even subtle costs that derive from its overall size and nine resistance genes accumulate in a prolonged experiment. This supports the line of reasoning where those plasmids that have accumulated large numbers of opportunistic genes are more dependent on specific selection on plasmid-carried genes than smaller plasmids ( 11 ). However, and similarly to pEC14pl1, RP4 also almost completely disappeared from all communities despite being relatively small (60 kb).…”

“…Mobilizable plasmids do not encode conjugation machineries but utilize the machineries encoded by other elements to hitchhike along the way and disperse from one cell to another ( 8 ). Among Gram-negative pathogens, conjugative and mobilizable plasmids are the most common genetic replicators mediating multidrug resistance ( 9 – 11 ).…”

Indirect Selection against Antibiotic Resistance via Specialized Plasmid-Dependent Bacteriophages

“…This is what we observe when isolated bacterial cultures are exposed to antimicrobials [ 2 ]. In practice and in environments where it has a notable effect (such as hospitals), however, bacteria most often receive a complete and efficient antibiotic resistance directly from their contemporaries instead of deciphering the evolutionary solution via mutations [ 4 , 5 ]. The resistance mechanism itself is often different from gradually developed ones, such as enzymes that hydrolyze antibiotic molecules [ 6 ].…”

“…When antibiotics are present, the eye of selection fixes on those who are not masked by a resistance. Yet, it is alerting that a single conjugative plasmid (such as PA1705-NDM) can carry resistance genes against most if not all antibiotics [ 5 ]. Therefore, in an environment where only a single antibiotic is present, a plasmid that provides resistance to a variety of antibiotics can become dominant.…”

“…Therefore, in an environment where only a single antibiotic is present, a plasmid that provides resistance to a variety of antibiotics can become dominant. In other words, nearly complete evolution of resistance to all drugs can occur in an instant even if such an evolutionary jump would appear completely impossible in a test tube [5]. Resistance providing genes may be transferred along with transposing elements from chromosomes to plasmids and vice versa.…”