Graphical abstract, Open Access
The hallmark of chronic infections is the difficulty in treating and curing infection with antibiotics. Biofilms are often the cause of chronic infections, due to their increased antibiotic tolerance and immune protective, aggregate structure. The antibiotic tolerance of biofilms is attributed mainly to the inability of the antibiotic to penetrate the biofilm, or the slow growth rate of bacteria in a biofilm. A recent study using a synthetic biology approach to examine the bacterial growth rate during an infection has provided some interesting results that challenge the current thinking regarding bacterial growth rates during infections.
In a brief report published in Cell, Host and Microbe, the team employs an engineered E. coli strain as a reporter for the bacterial growth rate during an infection model that mimics an orthopaedic device implanted into a bone that becomes infected. It is common during various medical procedures for the implant device or catheter to become colonized, and due to the inability to treat this infection, the device needs to be removed and reinserted.
Toggle switch = trigger and reporter elements
A toggle switch is an example of a synthetic biological circuit engineered in a cell to provide a reproducible and tuneable mechanism to turn on and off specific behaviours or reporters. In this case, the toggle switch is derepression of the TetR repressor upon exposure to the anhydrotetracycline (ATH) inducer, leading to induction of the Cro repressor. The Cro repressor now represses the cl regulator, both derived from the lambda cl/cro system, which ultimately leads to induction of the lacZ reporter. In the baseline state, lacZ is repressed, and the colony is white in the classic blue/white screen. During active growth in log phase, after 4 hrs of inducer treatment, all colonies switch to the lacZ expression state, producing blue colonies. The reporter works well in recapitulating the active growth state of log phase cultures, and the frequency of lacZ positive colonies decreases in non-growing or slow growth rates, such as the entry into stationary phase. Even with repression of the cl regulator, active cell division is still needed to dilute the active cl protein at the time of adding the tetracycline inducer. Therefore, the lacZ positive phenotype only occurs in actively dividing cells, but slow-growing cells will remain in the lacZ negative state.
The antibiotic levofloxacin, which kills actively dividing cells best, caused a decrease in cell number, and a decrease in the lacZ positive phenotype. Conversely, it does not kill stationary phase cells very well, and these cells had the lowest lacZ expression, all of which demonstrates further that the toggle switch is a reliable indicator of growth states.
Are bacteria in a slow growth, antibiotic tolerant state during an infection?
During chronic infections, it is proposed that treatment fails because it cannot accumulate to high enough concentrations in biofilms, or that bacteria are not actively growing, and therefore tolerant to antibiotic exposure. To test the latter of these two hypotheses, the E. coli reporter was used in this device-associated infection model, rather than Staphylococcus, the more typical cause of orthopaedic implant-related infections. The E. coli infection was maintained for 6 weeks, and after 4 days, about half of the bacterial cells in the infection are actively dividing, and half are not dividing. This was one of the first interesting results, which suggests that cells causing this infection do not mimic a stationary phase culture, a uniformly non-growing population. Then levofloxacin was used to treat the infection, and although bacterial load decreases compared to untreated infections (~1 log), it was predicted that the population of actively dividing cells would decrease. However, the opposite was shown, where the infection was enriched for actively dividing cells, which directly contrasts the killing data from in vitro experiments.
Were bacteria in the infection developing resistance? A few hundred isolates were recovered from the infection and none demonstrated any in vitro levofloxacin resistance. The authors concluded that the presence of non-dividing bacteria at the infection site does not lead to an antibiotic-tolerant infection. This conclusion directly contrasts what we know about the development of resistance under laboratory conditions, and highlights the utility of this kind of growth rate reporter during an actual infection. When the amount of antibiotic was increased, the infection could be cured, indicating that the non-dividing bacteria either begin to divide at some point, or an increase in concentration is enough to kill. The authors argue that either interpretation supports the unexpected observation that during infection, there are mixed growth rates of actively dividing and non-growing states, and that antibiotic resistance does not necessarily arise from the non-growing bacteria.
While the authors did not test the other tolerance hypothesis, that bacterial biofilms are the cause of chronic infections, it is assumed that biofilms formed on the inoculated pins. However, no studies were performed to confirm biofilm growth. Even in the presence of biofilms, these infections were cured with higher dose treatments, arguing perhaps that this was not necessarily a biofilm infection. This growth rate reporter would be interesting to examine under biofilm forming conditions, to test some of the underlying assumptions about heterogeneous growth rates in biofilms.
This straightforward, synthetic biology approach to design a reporter of the bacterial growth rate during an infection will be a useful tool in future antibiotic discovery research. Examining the bacterial response to antibiotics in a host system is ultimately more valuable than studying bacteria growing in a tube of broth.