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Due to the specific action on bacterial cell wall, β-lactam antibiotics have gained widespread usage as they exhibit a high degree of specificity in targeting bacteria, but causing minimal toxicity to host cells. Under antibiotic pressure, bacteria may opt to shed their cell walls and transform into L-form state as a means to evade the antibiotic effects. In this study, we explored and identified diverse optimal conditions for both Gram-negative bacteria (E. coli DH5α (CTX)) and Gram-positive bacteria (B. subtilis ATCC6633), which were induced to L-form bacteria using lysozyme (0.5 ppm) and meropenem (64 ppm). Notably, when bacteria transformed into L-form state, both bacterial strains showed varying degrees of increased resistance to antibiotics polymyxin E, meropenem, rifampicin, and tetracycline. E. coli DH5α (CTX) exhibited the most significant enhancement in resistance to tetracycline, with a 128-fold increase, while B. subtilis ATCC6633 showed a 32-fold increase in resistance to tetracycline and polymyxin E. Furthermore, L-form bacteria maintained their normal metabolic activity, combined with enhanced oxidative stress, served as an adaptive strategy promoting the sustained survival of L-form bacteria. This study provided a theoretical basis for comprehending antibiotic resistance mechanisms, developing innovative treatment strategies, and confronting global antibiotic resistance challenges.

[Display omitted] • L-forms were induced by lysozyme combined with meropenem. • The loss of the cell wall did not make bacteria less resistant to antibiotics. • L-form bacteria is not dormant subpopulation. • The division of L-forms is inhibited due to the block of the cell wall synthesis. • The stress response mechanism of bacteria helps the formation of L-form bacteria. Due to the specific action on bacterial cell wall, β-lactam antibiotics have gained widespread usage as they exhibit a high degree of specificity in targeting bacteria, but causing minimal toxicity to host cells. Under antibiotic pressure, bacteria may opt to shed their cell walls and transform into L-form state as a means to evade the antibiotic effects. In this study, we explored and identified diverse optimal conditions for both Gram-negative bacteria (E. coli DH5α (CTX)) and Gram-positive bacteria (B. subtilis ATCC6633), which were induced to L-form bacteria using lysozyme (0.5 ppm) and meropenem (64 ppm). Notably, when bacteria transformed into L-form state, both bacterial strains showed varying degrees of increased resistance to antibiotics polymyxin E, meropenem, rifampicin, and tetracycline. E. coli DH5α (CTX) exhibited the most significant enhancement in resistance to tetracycline, with a 128-fold increase, while B. subtilis ATCC6633 showed a 32-fold increase in resistance to tetracycline and polymyxin E. Furthermore, L-form bacteria maintained their normal metabolic activity, combined with enhanced oxidative stress, served as an adaptive strategy promoting the sustained survival of L-form bacteria. This study provided a theoretical basis for comprehending antibiotic resistance mechanisms, developing innovative treatment strategies, and confronting global antibiotic resistance challenges. [ABSTRACT FROM AUTHOR]

The rapid increase of circulating, antibiotic-resistant pathogens is a major ongoing global health crisis, and arguably, the end of the "golden age of antibiotics" is looming. This has led to a surge in research and development of alternative antimicrobials, including bacteriophages, to treat such infections (phage therapy). Isolating natural phage variants for the treatment of individual patients is an arduous and time-consuming task. Furthermore, the use of natural phages is frequently hampered by natural limitations, such as moderate in vivo activity, the rapid emergence of resistance, insufficient host range, or the presence of undesirable genetic elements within the phage genome. Targeted genetic editing of wild-type phages (phage engineering) has successfully been employed in the past to mitigate some of these pitfalls and to increase the therapeutic efficacy of the underlying phage variants. Clearly, there is a large potential for the development of novel, marker-less genome-editing methodologies to facilitate the engineering of therapeutic phages. Steady advances in synthetic biology have facilitated the in vitro assembly of modified phage genomes, which can be activated ("rebooted") upon transformation of a suitable host cell. However, this can prove challenging, especially in difficult-to-transform Gram-positive bacteria. In this chapter, we detail the production of cell wall-deficient L-form bacteria and their application to activate synthetic genomes of phages infecting Gram-positive host species., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)