Occurrence of cross-resistance and beta-lactam seesaw effect in glycopeptide, lipopeptide, and lipoglycopeptide-resistant MRSA correlates with membrane phosphatidylglycerol levels

ABSTRACTTreatment of methicillin-resistantStaphylococcus aureus(MRSA) infections is challenging and is associated with high rates of therapeutic failure. The glycopeptide (GP) vancomycin and the lipopeptide (LP) daptomycin are still relied upon to manage invasive MRSA infections; however, resistance to these antibiotics has emerged and there is evidence of cross-resistance between them. It has been observed that the susceptibility of MRSA to beta-lactams increases as susceptibility to GPs and LPs decreases, a phenomenon termed the seesaw effect. Recent efforts to understand the mechanism underlying the seesaw effect have focused on the penicillin binding proteins (PBPs). However, while daptomycin resistance is largely mediated by remodeling of membrane lipid composition, the role of membrane lipids in producing cross-resistance and the seesaw effect has not yet been investigated. Here, we evaluate the lipid profiles, cross susceptibilities, and beta-lactam susceptibilities of a collection of isogenic MRSA strains selected against daptomycin, vancomycin or dalbavancin (a lipoglycopeptide; LGP) to assess the relationship between membrane composition, cross-resistance, and the seesaw effect. We found that modification of membrane composition occurs not only in daptomycin-selected strains, but also vancomycin- and dalbavancin-selected strains. Significantly, we observed that typically the levels of short-chain phosphatidylglycerols (PGs) negatively correlate with MICs of GP/LP/LGP and positively correlate with MIC of certain beta-lactams, the latter being dependent on the primary PBP target of the particular beta-lactam. Furthermore, changes to certain PGs with long-chain fatty acids correlate well with presence of the seesaw effect. These studies demonstrate a major association between membrane remodeling and the seesaw effect.

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Occurrence of cross-resistance and β-lactam seesaw effect in glycopeptide-, lipopeptide- and lipoglycopeptide-resistant MRSA correlates with membrane phosphatidylglycerol levels

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz562 ◽

2020 ◽

Vol 75 (5) ◽

pp. 1182-1186 ◽

Cited By ~ 1

Author(s):

Kelly M Hines ◽

Tianwei Shen ◽

Nathaniel K Ashford ◽

Adam Waalkes ◽

Kelsi Penewit ◽

...

Keyword(s):

Cell Wall ◽

Susceptibility Testing ◽

Membrane Lipid ◽

Cross Resistance ◽

Membrane Composition ◽

Membrane Lipid Composition ◽

Usa300 Strain ◽

Associated Proteins ◽

The Relationship

Abstract Background Glycopeptides (GPs), lipopeptides (LPs) and lipoglycopeptides (LGPs) are related antimicrobials important for the management of invasive MRSA infections. Cross-resistance among these antibiotics in MRSA is well documented, as is the observation that susceptibility of MRSA to β-lactams increases as susceptibility to GPs and LPs decreases (i.e. the seesaw effect). Efforts to understand the relationship between GP/LP/LGP cross-resistance and the seesaw effect have focused on the PBPs, but the role of lipid metabolism has not been investigated. Objectives Since the cell membrane is structurally and metabolically integrated with the cell wall and anchors associated proteins, including PBPs, we examined the relationship between membrane lipid composition and the phenomena of cross-resistance among GPs/LPs/LGPs and the β-lactam seesaw effect. Methods We selected for daptomycin, vancomycin and dalbavancin resistance using the USA300 strain JE2 and evaluated the resulting mutants by WGS, MS-based lipidomics and antimicrobial susceptibility testing to assess the relationship between membrane composition, cross-resistance, and the seesaw effect. Results We observed cross-resistance to GPs/LPs/LGPs among the selected strains and the seesaw effect against various β-lactams, depending on the PBP targets of the particular β-lactam. We found that modification of membrane composition occurs not only in daptomycin-selected strains, but also vancomycin- and dalbavancin-selected strains. Significantly, we observed that the abundance of most phosphatidylglycerols positively correlates with MICs of GPs/LPs/LGPs and negatively correlates with the MICs of β-lactams. Conclusions These studies demonstrate a major association between membrane remodelling, cross-resistance and the seesaw effect.

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Synergy Between Beta-Lactams and Lipo-, Glyco-, and Lipoglycopeptides, Is Independent of the Seesaw Effect in Methicillin-Resistant Staphylococcus aureus

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.688357 ◽

2021 ◽

Vol 8 ◽

Author(s):

Rutan Zhang ◽

Ismael A. Barreras Beltran ◽

Nathaniel K. Ashford ◽

Kelsi Penewit ◽

Adam Waalkes ◽

...

Keyword(s):

Synergistic Effects ◽

Clinical Importance ◽

Synergistic Activity ◽

Beta Lactam ◽

Beta Lactams ◽

Methicillin Resistant ◽

Penicillin Binding Proteins ◽

The Impact ◽

Isogenic Strains

Methicillin-resistant S. aureus (MRSA) are resistant to beta-lactams, but synergistic activity between beta-lactams and glycopeptides/lipopeptides is common. Many have attributed this synergy to the beta-lactam-glycopeptide seesaw effect; however, this association has not been rigorously tested. The objective of this study was to determine whether the seesaw effect is necessary for synergy and to measure the impact of beta-lactam exposure on lipid metabolism. We selected for three isogenic strains with reduced susceptibility to vancomycin, daptomycin, and dalbavancin by serial passaging the MRSA strain N315. We used whole genome sequencing to identify genetic variants that emerged and tested for synergy between vancomycin, daptomycin, or dalbavancin in combination with 6 beta-lactams with variable affinity for staphylococcal penicillin binding proteins (PBPs), including nafcillin, meropenem, ceftriaxone, ceftaroline, cephalexin, and cefoxitin, using time-kills. We observed that the seesaw effect with each beta-lactam was variable and the emergence of the seesaw effect for a particular beta-lactam was not necessary for synergy between that beta-lactam and vancomycin, daptomycin, or dalbavancin. Synergy was more commonly observed with vancomycin and daptomycin based combinations than dalbavancin in time-kills. Among the beta-lactams, cefoxitin and nafcillin were the most likely to exhibit synergy using the concentrations tested, while cephalexin was the least likely to exhibit synergy. Synergy was more common among the resistant mutants than the parent strain. Interestingly N315-D1 and N315-DAL0.5 both had mutations in vraTSR and walKR despite their differences in the seesaw effect. Lipidomic analysis of all strains exposed to individual beta-lactams at subinhibitory concentrations suggested that in general, the abundance of cardiolipins (CLs) and most free fatty acids (FFAs) positively correlated with the presence of synergistic effects while abundance of phosphatidylglycerols (PGs) and lysylPGs mostly negatively correlated with synergistic effects. In conclusion, the beta-lactam-glycopeptide seesaw effect and beta-lactam-glycopeptide synergy are distinct phenomena. This suggests that the emergence of the seesaw effect may not have clinical importance in terms of predicting synergy. Further work is warranted to characterize strains that don’t exhibit beta-lactam synergy to identify which strains should be targeted with combination therapy and which ones cannot and to further investigate the potential role of CLs in mediating synergy.

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Target for bacteriostatic and bactericidal activities of beta-lactam antibiotics against Escherichia coli resides in different penicillin-binding proteins.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.39.4.812 ◽

1995 ◽

Vol 39 (4) ◽

pp. 812-818 ◽

Cited By ~ 29

Author(s):

G Satta ◽

G Cornaglia ◽

A Mazzariol ◽

G Golini ◽

S Valisena ◽

...

Keyword(s):

Escherichia Coli ◽

Penicillin Binding Protein ◽

Beta Lactam ◽

Beta Lactams ◽

Killing Effect ◽

Penicillin Binding Proteins ◽

Beta Lactam Antibiotics ◽

Bactericidal Activities ◽

The Given ◽

The Relationship

The relationship between cell-killing kinetics and penicillin-binding protein (PBP) saturation has been evaluated in the permeability mutant Escherichia coli DC2 in which the antimicrobial activity of beta-lactams has been described as being directly related to the extent of saturation of the PBP target(s). Saturation of a single PBP by cefsulodin (PBP 1s), mecillinam (PBP 2), and aztreonam (PBP 3) resulted in a slow rate of killing (2.5-, 1.5-, and 0.8-log-unit decreases in the number of CFU per milliliter, respectively, in 6 h). Saturation of two of the three essential PBPs resulted in a marked increase in the rate of killing, which reached the maximum value when PBPs 1s and 2 were simultaneously saturated by a combination of cefsulodin and mecillinam (4.7-log-unit decrease in the number of CFU per milliliter in 6 h). Inactivation of all three essential PBPs by the combination of cefsulodin, mecillinam, and aztreonam further increased the killing kinetics (5.5-log-unit decrease in the number of CFU per milliliter), and this was not significantly changed upon additional saturation of the nonessential PBPs 5 and 6 by cefoxitin. Similar relationships between PBP saturation and killing kinetics were obtained with imipenem and meropenem at concentrations which inhibited only one PBP (PBP 2), only two PBPs (PBP 1s and 2), or all three essential PBPs. Saturation of one or more PBPs also resulted in a different rate of bacteriolysis, the highest rate being obtained by the cefsulodin-mecillinam combination and by 5 micrograms of either imipenem or meropenem per ml. All of these conditions caused saturation of PBP 2 and saturation or extensive binding of PBP 1s. However, none of these conditions caused determined the fastest possible rate of killing, which occurred only when all three essential PBPs were saturated. It was concluded that the actual killing effect of beta-lactams is reflected by killing rates that approach the fastest possible rate for the given microorganism and that the targets for the bactericidal activity are precisely those PBPs whose saturation or binding occurs under conditions.

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Geometrical reorganization of Dectin-1 and TLR2 on single phagosomes alters their synergistic immune signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909870116 ◽

2019 ◽

Vol 116 (50) ◽

pp. 25106-25114 ◽

Cited By ~ 4

Author(s):

Wenqian Li ◽

Jun Yan ◽

Yan Yu

Keyword(s):

Immune Responses ◽

Immune Regulation ◽

Spatial Organization ◽

Innate Immune ◽

Innate Immune Responses ◽

Membrane Composition ◽

Intracellular Compartments ◽

Synergistic Regulation ◽

The Relationship

Receptors of innate immune cells function synergistically to detect pathogens and elicit appropriate immune responses. Many receptor pairs also appear “colocalized” on the membranes of phagosomes, the intracellular compartments for pathogen ingestion. However, the nature of the seemingly receptor colocalization and the role it plays in immune regulation are unclear, due to the inaccessibility of intracellular phagocytic receptors. Here, we report a geometric manipulation technique to directly probe the role of phagocytic receptor “colocalization” in innate immune regulation. Using particles with spatially patterned ligands as phagocytic targets, we can decouple the receptor pair, Dectin-1 and Toll-like receptor (TLR)2, to opposite sides on a single phagosome or bring them into nanoscale proximity without changing the overall membrane composition. We show that Dectin-1 enhances immune responses triggered predominantly by TLR2 when their centroid-to-centroid proximity is <500 nm, but this signaling synergy diminishes upon receptor segregation beyond this threshold distance. Our results demonstrate that nanoscale proximity, not necessarily colocalization, between Dectin-1 and TLR2 is required for their synergistic regulation of macrophage immune responses. This study elucidates the relationship between the spatial organization of phagocytic receptors and innate immune responses. It showcases a technique that allows spatial manipulation of receptors and their signal cross-talk on phagosomes inside living cells.

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Morphological deconvolution of beta-lactam polyspecificity inE. coli

10.1101/545335 ◽

2019 ◽

Author(s):

William J. Godinez ◽

Helen Chan ◽

Imtiaz Hossain ◽

Cindy Li ◽

Srijan Ranjitkar ◽

...

Keyword(s):

Ex Situ ◽

Exact Nature ◽

Enzyme Specificity ◽

Beta Lactam ◽

Beta Lactams ◽

Penicillin Binding Proteins ◽

E Coli ◽

Beta Lactam Antibiotics ◽

Cell Extracts ◽

The Family

AbstractBeta-lactam antibiotics comprise one of the earliest known classes of antibiotic therapies. These molsecules covalently inhibit enzymes from the family of penicillin-binding proteins, which are essential to the construction of the bacterial cell wall. As a result, beta-lactams have long been known to cause striking changes to cellular morphology. The exact nature of the changes tend to vary by the precise PBPs engaged in the cell since beta-lactams exhibit a range of PBP enzyme specificity. The traditional method for exploring beta-lactam polyspecificity is a gel-based binding assay which is low-throughput and typically runex situin cell extracts. Here, we describe a medium-throughput, image-based assay combined with machine learning methods to automatically profile the activity of beta-lactams inE. colicells. By testing for morphological change across a panel of strains with perturbations to individual PBP enzymes, our approach automatically and quantifiably relates different beta-lactam antibiotics according to their preferences for individual PBPs in cells. We show the potential of our approach for guiding the design of novel inhibitors towards different PBP-binding profiles by recapitulating the activity of two recently-reported PBP inhibitors.

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Role of efflux pump inhibitor in decreasing antibiotic cross-resistance of Pseudomonas aeruginosa in a burn hospital in Iran

The Journal of Infection in Developing Countries ◽

10.3855/jidc.7619 ◽

2016 ◽

Vol 10 (06) ◽

pp. 600-604 ◽

Cited By ~ 2

Author(s):

Mahshid Talebi-Taher ◽

َAli Majidpour ◽

Abbas Gholami ◽

Samira Rasouli-Kouhi ◽

Maryam Adabi

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Efflux Pumps ◽

Disk Diffusion ◽

Diffusion Method ◽

Cross Resistance ◽

Beta Lactams ◽

Efflux Pump Inhibitor ◽

Efflux Pump Inhibitors

Introduction: Multidrug resistance in Pseudomonas aeruginosa may be due to efflux pump overexpression. This study phenotypically examined the role of efflux pump inhibitors in decreasing antibiotic cross-resistance between beta-lactams, fluoroquinolones, and aminoglycosides in P. aeruginosa isolates from burn patients in Iran. Methodology: A total of 91 phenotypically and genotypically confirmed P. aeruginosa samples were studied. Multidrug cross-resistance was determined using the disk diffusion method and minimum inhibitory concentration (MIC) test. The contribution of efflux pumps was determined by investigating MIC reduction assay to markers of beta-lactams, fluoroquinolones, and aminoglycosides in the absence and presence of an efflux pump inhibitor. All the isolates were also tested by polymerase chain reaction for the presence of mexA, mexC, and mexE efflux genes. Results: Of the isolates, 81 (89%) and 83 (91.2%) were multidrug resistant according to the disk diffusion and MIC method, respectively. Cross-resistance was observed in 67 (73.6%) and 68 (74.7%) of isolates according to the disk diffusion and MIC method, respectively. In the presence of the efflux pump inhibitor, twofold or higher MIC reduction to imipenem, cefepime, ciprofloxacin, and gentamicin was observed in 59, 65, 55, and 60 isolates, respectively. Except for two isolates that were negative for mexC, all isolates were positive for mexA, mexC, and mexE genes simultaneously. Conclusion: Efflux pumps could cause different levels of resistance based on their expression in clinical isolates. Early detection of different efflux pumps in P. aeruginosa could allow the use of other antibiotics and efflux pump inhibitors in combination with antibiotic therapy.

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ThemecAHomologmecCConfers Resistance against β-Lactams in Staphylococcus aureus Irrespective of the Genetic Strain Background

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02731-13 ◽

2014 ◽

Vol 58 (7) ◽

pp. 3791-3798 ◽

Cited By ~ 31

Author(s):

Britta Ballhausen ◽

André Kriegeskorte ◽

Nina Schleimer ◽

Georg Peters ◽

Karsten Becker

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistance ◽

Regulatory System ◽

Wild Type ◽

Beta Lactam ◽

Beta Lactams ◽

Content Type ◽

Genetic Strain ◽

Mrsa Strains ◽

Strain Background

ABSTRACTIn staphylococci, methicillin resistance is mediated bymecA-encoded penicillin-binding protein 2a (PBP2a), which has a low affinity for beta-lactams. Recently, a novel PBP2a homolog was described as being encoded bymecC, which shares only 70% similarity tomecA. To prove thatmecCis the genetic determinant that confers methicillin resistance inStaphylococcus aureus, amecCknockout strain was generated. TheS. aureusΔmecCstrain showed considerably reduced oxacillin and cefoxitin MICs (0.25 and 4 μg/ml, respectively) compared to those of the corresponding wild-type methicillin-resistantS. aureus(MRSA) strain (8 and 16 μg/ml, respectively). Complementing the mutant intranswith wild-typemecCrestored the resistance to oxacillin and cefoxitin. By expressingmecCandmecAin differentS. aureusclonal lineages, we found thatmecCmediates resistance irrespective of the genetic strain background, yielding oxacillin and cefoxitin MIC values comparable to those withmecA. In addition, we showed thatmecCexpression is inducible by oxacillin, which supports the assumption that a functional beta-lactam-dependent regulatory system is active in MRSA strains possessing staphylococcal cassette chromosomemec(SCCmec) type XI. In summary, we showed thatmecCis inducible by oxacillin and mediates beta-lactam resistance in SCCmectype XI-carrying strains as well as in differentS. aureusgenetic backgrounds. Furthermore, our results could explain the comparatively low MICs for clinicalmecC-harboringS. aureusisolates.

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Altered permeability and beta-lactam resistance in a mutant of Mycobacterium smegmatis.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.8.1721 ◽

1997 ◽

Vol 41 (8) ◽

pp. 1721-1724 ◽

Cited By ~ 10

Author(s):

S Mukhopadhyay ◽

P Chakrabarti

Keyword(s):

Mycobacterium Smegmatis ◽

Parent Strain ◽

Beta Lactamase ◽

Wild Type ◽

Beta Lactam ◽

Beta Lactams ◽

Intact Cells ◽

Glycine Uptake ◽

Penicillin Binding Proteins ◽

High Level

Beta-lactam resistance in mycobacteria results from an interplay between the following: (i) beta-lactamase production, (ii) affinity of the penicillin-binding proteins (PBPs) for the drugs, and (iii) permeation of the drugs. A laboratory mutant of Mycobacterium smegmatis was studied in order to evaluate the roles of these factors in beta-lactam resistance. Mutant M13 was between 7- and 78-fold more resistant than the wild type to cephaloridine, cefoxitin, cefazolin, cefamandole, and cephalothin. Increased beta-lactamase activity toward these antibiotics was not observed in the mutant. The PBP profiles of the wild type and M13 were comparable. However, the affinities of PBP 1 for the beta-lactams tested were lower for the mutant than for the wild type. The permeation of the drugs measured in intact cells was lower for M13 than for the parent strain. The liposome swelling technique, which could be used for cephaloridine, also supported this view. Reduced permeation was not restricted to the beta-lactams alone. Glycine uptake was also lower in M13. Taken together, the results suggest that decreased affinities of PBP 1 for beta-lactams, combined with the decreased permeability of the cell wall of the mutant, lead to the development of high-level acquired beta-lactam resistance.

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Kinetics of penicillin binding to penicillin-binding proteins of Staphylococcus aureus

Biochemical Journal ◽

10.1042/bj3010139 ◽

1994 ◽

Vol 301 (1) ◽

pp. 139-144 ◽

Cited By ~ 42

Author(s):

H F Chambers ◽

M J Sachdeva ◽

C J Hackbarth

Keyword(s):

Staphylococcus Aureus ◽

Binding Proteins ◽

Beta Lactam ◽

Penicillin Binding Proteins ◽

Structural Modifications ◽

Drug Concentrations ◽

Lactam Antibiotic ◽

Resistant Strains ◽

The Relationship ◽

Kinetics Of

Reduced affinity of penicillin-binding proteins (PBPs) for binding penicillin has been proposed as a mechanism of beta-lactam antibiotic resistance in staphylococci. Penicillin binding by PBPs of three penicillin-susceptible and two penicillin-resistant strains of Staphylococcus aureus was studied in kinetic assays to determine rate constants, drug concentrations at which PBPs were bound and the relationship between concentrations that bound PBPs and concentrations that inhibited bacterial growth. PBPs 1 and 2 of the resistant strains exhibited slower acylation and more rapid deacylation than susceptible strains. In contrast PBP 4, a naturally low-affinity PBP, was modified such that it exhibited a lower rate of deacylation. The concentrations of penicillin at which modified PBPs were bound correlated with concentrations that inhibited growth of the resistant strains. Acquisition of penicillin resistance in these strains of S. aureus results, at least in part, from structural modifications affecting binding of multiple PBPs and appears to include recruitment of a non-essential PBP, PBP 4.

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The role of mprF mutations in “see-saw effect” of Daptomycin-resistant methicillin-resistant Staphylococcus aureus isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01295-21 ◽

2021 ◽

Author(s):

Shengnan Jiang ◽

Hemu Zhuang ◽

Feiteng Zhu ◽

Xiang Wei ◽

Junxiong Zhang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Novel Mutation ◽

Point Mutations ◽

Synergistic Activity ◽

Beta Lactam ◽

Beta Lactams ◽

Methicillin Resistant ◽

Beta Lactam Antibiotics ◽

Mrsa Strains ◽

The Impact

The emergence of daptomycin-resistant (DAP-R) Staphylococcus aureus strains has become a global problem. Point mutations in mprF are the main cause of daptomycin (DAP) treatment failure. However, the impact of these specific point-mutations in methicillin-resistant S. aureus (MRSA) strains associated with DAP resistance and the “see-saw effect” of distinct beta-lactams remains unclear. In this study, we used three series of clinical MRSA strains with three distinct mutated mprF alleles from clone complexes (CC) 5 and 59 to explore the “see-saw effect” and the combination effect of DAP plus beta-lactams. Through construction of mprF deletion and complementation strains of SA268, we determined that mprF -S295A, mprF -S337L and one novel mutation of mprF- I348del within the bifunctional domain lead to DAP resistance. Compared with wild-type mprF cloned from a DAP-susceptible (DAP-S) strain, these three mprF mutations conferred the “see-saw effect” to distinct beta-lactams in the SA268Δ mprF strains and mutated- mprF (I348del and S337L) did not alter the cell surface positive charge ( P > 0.05). The susceptibility to beta-lactams increased significantly in DAP-R CC59 strains and the “see-saw effect” was found to be associated with distinct mutated mprF alleles and the category of beta-lactams. The synergistic activity of DAP plus oxacillin was detected in all DAP-R MRSA strains. Continued progress in understanding the mechanism of restoring susceptibility to beta-lactam antibiotics mediated by the mprF mutation and its impact on beta-lactam combination therapy will provide fundamental insights into treatment of MRSA infections.

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