The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.S. infecting a single Arthrobacter sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp) to over 70 kbp, and G+C contents range from 45–68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these Arthrobacter phages are primarily lytic, and only the singleton Galaxy is likely temperate.

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.

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

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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...