Biology subjects

Cross, T.

Publications and source records attributed to Cross, T..

3 recordsLinked to original sources

bioRxiv2025

Prevalence and mechanisms of high-level carbapenem antibiotic tolerance in clinical isolates of Klebsiella pneumoniae

Antibiotic tolerance is the ability of bacteria to survive normally lethal doses of antibiotics for extended time periods. Clinically significant Enterobacterales, for example, can exhibit high tolerance to the last-resort antibiotic meropenem. Meropenem tolerance is associated with formation of cell wall-deficient spheroplasts that readily recover to rod shape and normal growth upon removal of the antibiotic. Both the true prevalence of tolerance, and genetic mechanisms underlying it, remain poorly understood. Here, we find that meropenem tolerance is widespread among clinical Enterobacterales. Using forward genetics, we uncover novel tolerance factors in a hypertolerant isolate of the ESKAPE pathogen Klebsiella pneumoniae. We find that multiple mechanisms contribute to tolerance, and that cell envelope stress responses (PhoPQ, Cpx, Rcs and OmpR/EnvZ) collectively promote spheroplast stability and recovery, while the lytic transglycosylase MltB counteracts it. Our data indicate that tolerance is widespread among clinical isolates, and that outer membrane maintenance is a key factor promoting survival of tolerant K. pneumoniae.

microbiology↗

bioRxiv2021

The PhoPQ two-component system promotes carbapenem tolerance in Enterobacterales.

Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of {beta}-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface. How a membrane structure like LPS, with its reliance on mere electrostatic interactions to maintain stability, is capable of countering internal turgor pressure is unknown. Here, we have uncovered a novel role for the PhoPQ two-component system in tolerance to the {beta}-lactam antibiotic meropenem in Enterobacterales. We found that PhoPQ is induced by meropenem treatment and promotes an increase in 4-amino-4-deoxy-L-aminoarabinose [L-Ara4N] modification of lipid A, the membrane anchor of LPS. L-Ara4N modifications enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulatory element mgrB (commonly selected for during clinical therapy with the last-resort antibiotic colistin, an antimicrobial peptide [AMP]) results in dramatically enhanced tolerance, suggesting that AMPs can collaterally select for meropenem tolerance via stable overactivation of PhoPQ. Lastly, we identify histidine kinase inhibitors (including an FDA-approved drug) that inhibit PhoPQ-dependent LPS modifications and consequently potentiate meropenem to enhance killing of tolerant cells. In summary, our results suggest that PhoPQ-mediated LPS modifications play a significant role in stabilizing the OM, promoting survival when the primary integrity maintenance structure, the cell wall, is removed. SignificanceTreating an infection with an antibiotic often fails, resulting in a tremendous public health burden. One understudied likely reason for treatment failure is the development of "antibiotic tolerance", the ability of bacteria to survive normally lethal exposure to an antibiotic. Here, we describe a molecular mechanism promoting tolerance. A bacterial stress sensor (PhoPQ) is activated in response to antibiotic (meropenem) treatment and consequently strengthens a bacterial protective "shell" to enhance survival. We also identify inhibitors of this mechanism, opening the door to developing compounds that help antibiotics work better against tolerant bacteria.

microbiology↗

bioRxiv2019

Spheroplast-mediated carbapenem tolerance in Gram-negative pathogens

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied, yet likely widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae is able to tolerate exposure to the typically bactericidal {beta}-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread tolerance is. Here, we have 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 pneumoniae, and Klebsiella aerogenes, but not Escherichia coli, exhibit moderate to high levels of tolerance to meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered to wild-type morphology and exponential 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.

microbiology↗