Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal β-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread β-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

Summary The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a covalently closed network of peptidoglycan (PG) strands. While PG synthesis is important for survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by ‘autolysins’, enzymes that break down PG. Cell division, for example, requires extensive PG remodeling, especially during separation of daughter cells, which depends heavily upon the activity of amidases. However, in Vibrio cholerae, we demonstrate that amidase activity alone is insufficient for daughter cell separation and that lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low‐salt media. The division‐specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum cleave PG strands that are crosslinked beyond the reach of the highly regulated activity of the amidase and clear PG debris that may block the completion of outer membrane invagination.

24Antibiotic tolerance, the ability to temporarily sustain viability in the presence of 25 bactericidal antibiotics, constitutes an understudied, yet likely widespread cause of 26 antibiotic treatment failure. We have previously shown that the Gram-negative pathogen 27Vibrio cholerae is able to tolerate exposure to the typically bactericidal -lactam antibiotics 28 by assuming a spherical morphotype devoid of detectable cell wall material. However, it 29 is unclear how widespread tolerance is. Here, we have tested a panel of clinically 30 significant Gram-negative pathogens for their response to the potent, broad-spectrum 31 carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter 32 cloacae, Klebsiella pneumoniae, and Klebsiella aerogenes, but not Escherichia coli, 33 exhibit moderate to high levels of tolerance to meropenem, both in laboratory growth 34 medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient 35 spheroplasts, which readily recovered to wild-type morphology and exponential growth 36 upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in 37 clinically significant bacterial species, and we suggest that this could contribute to 38 treatment failure associated with these organisms. 39 40 41 42 43 44 45 possession of the carbapenemase KPC (Klebsiella pneumoniae carbapenemase). 132 133 Among the susceptible/non-resistant, non-carbapenemase producing isolates, killing and 134optical density dynamics varied widely between species and even isolates within the 135 same species (e.g., E. cloacae WCM0001 versus E. cloacae ARB0008) ( Fig. 1). 136Interestingly, both in lysis behavior and survival, E. coli was considerably less tolerant

18Bactericidal antibiotics are powerful drugs due to their ability to not only inhibit essential 19 bacterial functions, but to convert them into toxic (and potentially lethal) processes. 20However, many important bacterial pathogens are remarkably tolerant against 21 bactericidal drugs, due to inducible stress responses that repair antibiotic-induced 22 damage. The mechanistic details of how stress responses promote whole population 23 tolerance in important human pathogens are unknown. The two-component system 24 VxrAB of the diarrheal pathogen Vibrio cholerae, a model system for high-level -lactam 25 2 tolerance, is induced by exposure to cell wall acting antibiotic and controls a gene 26 network encoding highly diverse functions, including cell wall synthesis functions and 27 iron uptake systems. Here, we show that positive control over cell wall synthesis 28 functions only partially explains high level -lactam tolerance. We find that in addition to 29 cell wall damage, -lactam antibiotics inappropriately induce the Fur-regulated iron 30 starvation response, causing an increase in intracellular free iron levels and colateral 31 oxidative damage. We propose that VxrAB reduces antibiotic-induced toxic influx of 32 Fe 2+ and concomitant metabolic perturbations by selectively downregulating iron uptake 33 transporters. Our results suggest that the ability to counteract diverse antibiotic-induced 34 stresses promotes high-level antibiotic tolerance and highlight the complex responses 35 elicited by antibiotics in addition to their primary mechanism of action. 36 37 by either developing the ability to grow in their presence (antibiotic resistance, ABR) or 49 to simply stay alive in their presence for extended time periods (antibiotic 50 tolerance/persistence) [3][4][5][6][7][8] . While the mechanisms and consequences of ABR are 51 relatively well-established, antibiotic tolerance remains poorly understood, limiting our 52 ability to develop antibiotic adjuvants that increase the efficacy of existing drugs. 53 54The -lactam antibiotics (penicillins, cephalosporins, carbapenems, cephamycins and 55 monobactams) are highly potent bactericidal agents. Their typically lethal action results 56 from their ability to simultaneously inhibit multiple targets (i.e, the transpeptidase 57 domain of multiple penicillin-binding proteins [PBPs]), which ultimately causes bacterial 58 cells to deplete essential cell wall precursors and self-destruct through the activity of 59 endogenous, cell wall lytic enzymes ('autolysins'; endopeptidases, amidases and lytic 60 transglycosylases) 9-12 . However, we and others have recently shown that many 61 clinically significant Gram-negative pathogens are remarkably -lactam tolerant. The 62 cholera pathogen Vibrio cholerae, the opportunistic pathogen Pseudomonas aeruginosa 63 and clinical isolates of Enterobacteriaceae all survive treatment with -lactam antibiotics 64 (including the "last resort" agent meropenem) by forming non-dividing, cell wall deficient 65 spheroplasts 11,13,14 . Upon...

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13The cell wall is a crucial structural feature in the vast majority of bacteria and 14 comprises a rigid, covalently closed, mesh-like network of peptidoglycan (PG) strands. 15While PG synthesis is important for bacterial survival under many conditions, the cell wall 16is also a dynamic structure, undergoing degradation and remodeling by so-called 17 "autolysins", enzymes that break bonds in the PG network. Cell division, for example, 18 requires extensive PG remodeling and separation of daughter cells, which depends 19 primarily upon the activity of amidases. However, in V. cholerae, we have found that 20 amidase activity alone is insufficient for daughter cell separation and that the lytic 21 transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both 22 amidase and to clear PG debris that may block the completion of outer-membrane 1 invagination. 2 3 191(13), 4186-4194. https://doi.