Synergism of imipenem with fosfomycin associated with the active cell wall recycling and heteroresistance in Acinetobacter calcoaceticus-baumannii complex

The carbapenem-resistant Acinetobacter calcoaceticus-baumannii (ACB) complex has become an urgent threat worldwide. Here, we determined antibiotic combinations and the feasible synergistic mechanisms against three couples of ACB (A. baumannii (AB250 and A10), A. pittii (AP1 and AP23), and A. nosocomialis (AN4 and AN12)). Imipenem with fosfomycin, the most effective in the time-killing assay, exhibited synergism to all strains except AB250. MurA, a fosfomycin target encoding the first enzyme in the de novo cell wall synthesis, was observed with the wild-type form in all isolates. Fosfomycin did not upregulate murA, indicating the MurA-independent pathway (cell wall recycling) presenting in all strains. Fosfomycin more upregulated the recycling route in synergistic strain (A10) than non-synergistic strain (AB250). Imipenem in the combination dramatically downregulated the recycling route in A10 but not in AB250, demonstrating the additional effect of imipenem on the recycling route, possibly resulting in synergism by the agitation of cell wall metabolism. Moreover, heteroresistance to imipenem was observed in only AB250. Our results indicate that unexpected activity of imipenem on the active cell wall recycling concurrently with the presence of heteroresistance subpopulation to imipenem may lead to the synergism of imipenem and fosfomycin against the ACB isolates.

A New Class of Cell Wall-Recycling l , d -Carboxypeptidase Determines β-Lactam Susceptibility and Morphogenesis in Acinetobacter baumannii

To grow efficiently, resist antibiotics, and control the immune response, bacteria recycle parts of their cell wall. A key step in the typical recycling pathway is the reuse of cell wall peptides by an enzyme known as an l , d -carboxypeptidase (LDC). Acinetobacter baumannii , an “urgent-threat” pathogen causing drug-resistant sepsis in hospitals, was previously thought to lack this enzymatic activity due to absence of a known LDC homolog.

A new class of cell wall-recycling L,D-carboxypeptidase determines β-lactam susceptibility and morphogenesis in Acinetobacter baumannii

The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for cell elongation and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell-wall-modifying L,D-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic L,D-carboxypeptidase (LDC) that provides a critical step in cell-wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, elongation, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including L,D-crosslink formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on cell envelope physiology. We thus reveal a novel class of cell-wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria.

An efficient synthesis of 1,6-anhydro-N-acetylmuramic acid from N-acetylglucosamine

A novel synthesis of 1,6-anhydro-N-acetylmuramic acid is described, which proceeds in only five steps from the cheap starting material N-acetylglucosamine. This efficient synthesis should enable future studies into the importance of 1,6-anhydromuramic acid in bacterial cell wall recycling processes.

Identification of MupP as a New Peptidoglycan Recycling Factor and Antibiotic Resistance Determinant in Pseudomonas aeruginosa

ABSTRACT Peptidoglycan (PG) is an essential cross-linked polymer that surrounds most bacterial cells to prevent osmotic rupture of the cytoplasmic membrane. Its synthesis relies on penicillin-binding proteins, the targets of beta-lactam antibiotics. Many Gram-negative bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, are resistant to beta-lactams because of a chromosomally encoded beta-lactamase called AmpC. In P. aeruginosa, expression of the ampC gene is tightly regulated and its induction is linked to cell wall stress. We reasoned that a reporter gene fusion to the ampC promoter would allow us to identify mutants defective in maintaining cell wall homeostasis and thereby uncover new factors involved in the process. A library of transposon-mutagenized P. aeruginosa was therefore screened for mutants with elevated ampC promoter activity. As an indication that the screen was working as expected, mutants with transposons disrupting the dacB gene were isolated. Defects in DacB have previously been implicated in ampC induction and clinical resistance to beta-lactam antibiotics. The screen also uncovered murU and PA3172 mutants that, upon further characterization, displayed nearly identical drug resistance and sensitivity profiles. We present genetic evidence that PA3172, renamed mupP, encodes the missing phosphatase predicted to function in the MurU PG recycling pathway that is widely distributed among Gram-negative bacteria. IMPORTANCE The cell wall biogenesis pathway is the target of many of our best antibiotics, including penicillin and related beta-lactam drugs. Resistance to these therapies is on the rise, particularly among Gram-negative species like Pseudomonas aeruginosa, a problematic opportunistic pathogen. To better understand how these organisms resist cell wall-targeting antibiotics, we screened for P. aeruginosa mutants defective in maintaining cell wall homeostasis. The screen identified a new factor, called MupP, involved in the recycling of cell wall turnover products. Characterization of MupP and other components of the pathway revealed that cell wall recycling plays important roles in both the resistance and the sensitivity of P. aeruginosa to cell wall-targeting antibiotics. IMPORTANCE The cell wall biogenesis pathway is the target of many of our best antibiotics, including penicillin and related beta-lactam drugs. Resistance to these therapies is on the rise, particularly among Gram-negative species like Pseudomonas aeruginosa, a problematic opportunistic pathogen. To better understand how these organisms resist cell wall-targeting antibiotics, we screened for P. aeruginosa mutants defective in maintaining cell wall homeostasis. The screen identified a new factor, called MupP, involved in the recycling of cell wall turnover products. Characterization of MupP and other components of the pathway revealed that cell wall recycling plays important roles in both the resistance and the sensitivity of P. aeruginosa to cell wall-targeting antibiotics.