## 5 Biofilm and Heterogeneity of _A. baumannii_ Populations
### Persisters and Heterogeneity of A. baumannii Populations  
Persisters are able to survive exposure to a bactericidal drug concentration, and they usually constitute a small fraction of bacterial populations [156]. Antibiotic persistence is a transient state, and, when persisters resume growth after drug treatment, their progeny become antibiotic susceptible. There is an ongoing debate about mechanisms underlying persister formation [157; 158; 159; 160]. It is well known that biofilms provide conducive niches that favor the formation of persisters [161]. Persisters can arise spontaneously or in response to stress caused by antibiotics, the host immune response, ROS, high osmolarity, pH changes, diauxic shift, desiccation, or nutrient starvation. In addition to the stringent response mentioned above, toxin-antitoxin modules, quorum signaling, efflux pumps, the SOS, and oxidative stress responses can be activated during persister formation. These pathways and stimuli may lead to decreased metabolism, depletion of ATP, protein aggregation, and inhibition of translation [162; 163; 132]. It was recently demonstrated that, in _E. coli_, (p)ppGpp induces production of the ribosome modulation factor (RMF), the hibernation-promoting factor (Hpf), and the ribosome-associated inhibitor (RaiA), which convert active 70S ribosomes into inactive 70S, 90S, and 100S ribosomes, leading to translation inhibition [164]. It should be noted that, due to diverse conditions in biofilm structures, multiple mechanisms triggering antibiotic tolerance, and stochastic effects, persister subpopulations should be considered as a heterogeneous group of cells. The formation of _A. baumannii_i persisters induced by polymixin B, meropenem, and ceftazidime was reported [165; 166; 167; 168]. It was also found that the ppGpp deficiency in the _A. baumannii__A_relA_ strain reduced formation of persister cells tolerant to colistin and rifampicin [127]. The analysis of the transcriptome of persisters tolerant to ceftazidime revealed upregulation of two toxin-antitoxin systems HigB/HigA and DUF1044/RelB, as well as downregulation of certain metabolic pathways, including the electron transport chain and citrate cycle [168]. Interestingly, the expression of genes associated with biodegradation pathways of aromatic compounds was detected in persistent cells. It was suggested that the degradation of aromatic rings in antibiotics could be utilized by _A. baumannii_i persisters during nutrient starvation [168]. [PERSON] et al. found that the major fraction of _A. baumannii_i persisters that survive \(\beta\)-lactam antibiotic treatment contains spherical non-walled, but metabolically active cells [170]. In contrast to wall-deficient, so-called L-forms of other Gram-negative bacteria,A. baumannii_ non-walled cells were able to survive without any osmoprotective agent. This type of _A. baumannii_ persister cell was also formed during antibiotic therapy in vivo in _Galeria melonella_ larvae which were used as the infection model. Biofilms provide a conducive environment facilitating not only phenotypic heterogeneity (e.g., persister formation) but also genetic diversification. It was demonstrated that evolution within _A. baumannii_ biofilms can generate greater genetic diversity than in planktonic, well-mixed populations [171]. Planktonic cells exposed to ciprofloxacin shared the same limited number of mutations in topoisomerase (the primary drug target), whereas biofilm-adapted populations acquired different types of mutations in the regulators of the efflux pumps. The emergence of a certain trade-off between fitness and resistance level was detected; biofilm-adapted clones were less drug-resistant than planktonic cells, but more fit in the absence of the drug [171]. Other studies demonstrated that the exposure of _A. baumannii_ biofilms to sub-inhibitory concentrations of ciprofloxacin or tetracycline led to the generation of genetic and phenotypic diversity among biofilm dispersal isolates [172].