Last month’s announcement of an mcr1-bearing plasmid in a U.S. patient isolate caused quite a stir, and for good reason. This gene confers resistance to colistin, an antibiotic of last resistance, and its existence on a plasmid means relatively easy transfer between bacterial types. The possibility (or probability, given drug-resistant infection history) of colistin resistance spreading among other bacteria is one of the reasons this story received so much attention.
Bacteria acquire and spread genetic information through several means. Some bacteria form a long, thin tube called a pilus, mediating transfer of DNA by direct cell-to-cell contact, in a process known as conjugation. Some bacterial take up environmental DNA and incorporate it into their existing genome in a process called transformation. This cell contact-independent transformation process requires the cells to be competent, or able to take up extracellular DNA. Some bacterial species can be induced under laboratory conditions to take up extracellular DNA; others are naturally competent dependent on a certain growth stage, nutrient, or other condition. With numbers of drug-resistant infections growing, scientists need to better understand how pathogenic microbes acquire and spread resistance genes.
Now new research available in Antimicrobial Agents and Chemotherapy has identified conditions that allow Acinetobacter baumannii, an increasingly common infectious microbe, to become competent for transformation. Infections with the Gram-negative A. baumannii are not only increasing in incidence, but are often resistant to antibiotic therapy. Genomic studies show that the bacterium can acquire foreign DNA relatively easily, and previous studies demonstrated that A. baumannii can be lab-induced to competency. First author German Matias Traglia, working with senior researcher Maria Soledad Ramirez, set to finding what induces natural competence in this species.
The first clue that competence may be induced by a host-related environmental signal came from the discovery that the optimal pH to induce competence is 7.5, similar to the pH of blood. The second clue came from testing transformation efficiency after bacterial growth in several different media. This showed that cells grown in Complete Transformation Medium (CTM) were fourfold more efficient at taking up and incorporating DNA than those grown in Luria-Bertani broth (LB). Two major components are found in CTM but not LB: calcium chloride and bovine serum albumin (BSA). Next, the team turned to testing these components.
Adding either BSA or CaCl2 to LB increased A. baumannii transformation efficiency, but only when both components were added simultaneously did they confer CTM-levels of transformation (see right). To confirm the role of CaCl2 in competence induction, the research team added EDTA, a calcium chelator, to the CTM media, effectively sequestering the calcium from the bacterium. The EDTA-treated CTM induced a lower transformation efficiency than untreated CTM (although still higher than LB, since BSA was still present in the EDTA-CTM). This systematic addition or removal of CaCl2 confirmed its role in A. baumannii natural transformation.
Further analysis of the role of albumin in transformation showed that both heat-treated (and thus denatured) or trypsin-treated (and thus fragmented) BSA were both able to induce A. baumannii competence. However, there is something specific about albumin required for the process, since other proteins, such as casein, didn’t have the same effect. Importantly, the authors showed that human serum albumin also induced competence, demonstrating that this process can occur during infection.
Why do BSA and CaCl2 increase natural transformation of A. baumannii? Addition of BSA or CaCl2 induced expression of competence-related genes comEA and pilQ, partially explaining how these components increase natural transformation efficiency (see left). BSA functions as a carrier in the bloodstream, and one of its cargo is calcium, meaning that A. baumannii will likely be exposed to both in the host. In this high-stress environment, it may be advantageous for the bacterium to take up foreign DNA that could provide a selective advantage. Extracellular DNA – perhaps released from biofilm matrix – could contain resistance genes, contributing to acquisition and spread of A. baumannii drug-resistant infections.
-- Julie Wolf