Perturbations of Phosphatidate Cytidylyltransferase (CdsA) Mediate Daptomycin Resistance in Streptococcus mitis/oralis by a Novel Mechanism

ABSTRACT Streptococcus mitis/oralis is an important pathogen, causing life-threatening infections such as endocarditis and severe sepsis in immunocompromised patients. The β-lactam antibiotics are the usual therapy of choice for this organism, but their effectiveness is threatened by the frequent emergence of resistance. The lipopeptide daptomycin (DAP) has been suggested for therapy against such resistant S. mitis/oralis strains due to its in vitro bactericidal activity and demonstrated efficacy against other Gram-positive pathogens. Unlike other bacteria, however, S. mitis/oralis has the unique ability to rapidly develop stable, high-level resistance to DAP upon exposure to the drug both in vivo and in vitro. Using isogenic DAP-susceptible and DAP-resistant S. mitis/oralis strain pairs, we describe a mechanism of resistance to both DAP and cationic antimicrobial peptides that involves loss-of-function mutations in cdsA (encoding a phosphatidate cytidylyltransferase). CdsA catalyzes the synthesis of cytidine diphosphate-diacylglycerol, an essential phospholipid intermediate for the production of membrane phosphatidylglycerol and cardiolipin. DAP-resistant S. mitis/oralis strains demonstrated a total disappearance of phosphatidylglycerol, cardiolipin, and anionic phospholipid microdomains from membranes. In addition, these strains exhibited cross-resistance to cationic antimicrobial peptides from human neutrophils (i.e., hNP-1). Interestingly, CdsA-mediated changes in phospholipid metabolism were associated with DAP hyperaccumulation in a small subset of the bacterial population, without any binding by the remaining larger population. Our results indicate that CdsA is the major mediator of high-level DAP resistance in S. mitis/oralis and suggest a novel mechanism of bacterial survival against attack by antimicrobial peptides of both innate and exogenous origins.

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Evidence for Inhibition of Topoisomerase 1A by Gold(III) Macrocycles and Chelates TargetingMycobacterium tuberculosisandMycobacterium abscessus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01696-17 ◽

2018 ◽

Vol 62 (5) ◽

Cited By ~ 6

Author(s):

Rashmi Gupta ◽

Carolina Rodrigues Felix ◽

Matthew P. Akerman ◽

Kate J. Akerman ◽

Cathryn A. Slabber ◽

...

Keyword(s):

Mycobacterium Abscessus ◽

Cross Resistance ◽

Human Pathogens ◽

Intrinsic Resistance ◽

Content Type ◽

Starting Point ◽

Scalable Synthesis ◽

High Level ◽

Novel Drug

ABSTRACTMycobacterium tuberculosisand the fast-growing speciesMycobacterium abscessusare two important human pathogens causing persistent pulmonary infections that are difficult to cure and require long treatment times. The emergence of drug-resistantM. tuberculosisstrains and the high level of intrinsic resistance ofM. abscessuscall for novel drug scaffolds that effectively target both pathogens. In this study, we evaluated the activity of bis(pyrrolide-imine) gold(III) macrocycles and chelates, originally designed as DNA intercalators capable of targeting human topoisomerase types I and II (Topo1 and Topo2), againstM. abscessusandM. tuberculosis. We identified a total of 5 noncytotoxic compounds active against both mycobacterial pathogens under replicatingin vitroconditions. We chose one of these hits, compound 14, for detailed analysis due to its potent bactericidal mode of inhibition and scalable synthesis. The clinical relevance of this compound was demonstrated by its ability to inhibit a panel of diverseM. tuberculosisandM. abscessusclinical isolates. Prompted by previous data suggesting that compound 14 may target topoisomerase/gyrase enzymes, we demonstrated that it lacked cross-resistance with fluoroquinolones, which target theM. tuberculosisgyrase.In vitroenzyme assays confirmed the potent activity of compound 14 against bacterial topoisomerase 1A (Topo1) enzymes but not gyrase. Novel scaffolds like compound 14 with potent, selective bactericidal activity againstM. tuberculosisandM. abscessusthat act on validated but underexploited targets like Topo1 represent a promising starting point for the development of novel therapeutics for infections by pathogenic mycobacteria.

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Efficacy of Biocides Used in the Modern Food Industry To Control Salmonella enterica, and Links between Biocide Tolerance and Resistance to Clinically Relevant Antimicrobial Compounds

Applied and Environmental Microbiology ◽

10.1128/aem.07534-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3087-3097 ◽

Cited By ~ 106

Author(s):

Orla Condell ◽

Carol Iversen ◽

Shane Cooney ◽

Karen A. Power ◽

Ciara Walsh ◽

...

Keyword(s):

Salmonella Enterica ◽

Food Industry ◽

Multidrug Resistant ◽

Cross Resistance ◽

Antimicrobial Compounds ◽

Cross Tolerance ◽

Content Type ◽

Food Grade ◽

High Level

ABSTRACTBiocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR)Salmonella entericastrains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds ofin vitroselection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure ofSalmonellastrains to an active biocidal compound, a high-level of tolerance was selected for a number ofSalmonellaserotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonicSalmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.

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EarlyIn VitroandIn VivoDevelopment of High-Level Daptomycin Resistance Is Common in Mitis Group Streptococci after Exposure to Daptomycin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01921-12 ◽

2013 ◽

Vol 57 (5) ◽

pp. 2319-2325 ◽

Cited By ~ 28

Author(s):

Cristina García-de-la-Mària ◽

Juan M. Pericas ◽

Ana del Río ◽

Ximena Castañeda ◽

Xavier Vila-Farrés ◽

...

Keyword(s):

Body Weight ◽

Streptococcus Bovis ◽

Streptococcus Mitis ◽

Content Type ◽

Group 4 ◽

Viridans Group Streptococcus ◽

High Level ◽

Time Kill

ABSTRACTThe development of high-level daptomycin resistance (HLDR; MIC of ≥256 mg/liter) after exposure to daptomycin has recently been reported in viridans group streptococcus (VGS) isolates. Our study objectives were as follows: to know whetherin vitrodevelopment of HLDR after exposure to daptomycin was common among clinical isolates of VGS andStreptococcus bovis; to determine whether HLDR also developed during the administration of daptomycin to treat experimental endocarditis caused by the daptomycin-susceptible, penicillin-resistantStreptococcus mitisstrainS. mitis351; and to establish whether combination with gentamicin prevented the development of HLDRin vitroandin vivo. In vitrostudies were performed with 114 VGS strains (mitis group, 92; anginosus group, 10; mutans group, 8; and salivarius group, 4) and 54Streptococcus bovisstrains isolated from 168 consecutive patients with infective endocarditis diagnosed between 1995 and 2010. HLDR was only observed after 24 h of exposure to daptomycin in 27% of the mitis group, including 27% ofS. mitisisolates, 47% ofS. oralisisolates, and 13% ofS. sanguisisolates. In our experimental model, HLDR was detected in 7/11 (63%) and 8/12 (67%) isolates recovered from vegetations after 48 h of daptomycin administered at 6 mg/kg of body weight/24 h and 10 mg/kg/24 h, respectively.In vitro, time-kill experiments showed that daptomycin plus gentamicin was bactericidal againstS. mitis351 at tested concentrations of 0.5 and 1 times the MIC and prevented the development of HLDR.In vivo, the addition of gentamicin at 1 mg/kg/8 h to both daptomycin arms prevented HLDR in 21 out of 23 (91%) rabbits. Daptomycin plus gentamicin was at least as effective as vancomycin plus gentamicin. In conclusion, HLDR develops rapidly and frequentlyin vitroandin vivoamong mitis group streptococci. Combining daptomycin with gentamicin enhanced its activity and prevented the development of HLDR in most cases.

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Impaired Parasite Attachment as Fitness Cost of Metronidazole Resistance in Giardia lamblia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00384-11 ◽

2011 ◽

Vol 55 (10) ◽

pp. 4643-4651 ◽

Cited By ~ 36

Author(s):

Noa Tejman-Yarden ◽

Maya Millman ◽

Tineke Lauwaet ◽

Barbara J. Davids ◽

Frances D. Gillin ◽

...

Keyword(s):

Glucose Metabolism ◽

Cell Lines ◽

Giardia Lamblia ◽

Cross Resistance ◽

Content Type ◽

Metronidazole Resistance ◽

High Level ◽

Plastic Surfaces

ABSTRACTInfections with the diarrheagenic protozoan pathogenGiardia lambliaare most commonly treated with metronidazole (Mz). Treatment failures with Mz occur in 10 to 20% of cases and Mz resistance develops in the laboratory, yet clinically, Mz-resistant (Mzr)G. lambliahas rarely been isolated from patients. To understand why clinical Mzrisolates are rare, we questioned whether Mz resistance entails fitness costs to the parasite. Our studies employed several newly generated and established isogenic Mzrcell lines with stable, high-level resistance to Mz and significant cross-resistance to tinidazole, nitazoxanide, and furazolidone. Oral infection of suckling mice revealed that three of five Mzrcell lines could not establish infection, while two Mzrcell lines infected pups, albeit with reduced efficiencies. Failure to colonize resulted from a diminished capacity of the parasite to attach to the intestinal mucosain vivoand to epithelial cells and plastic surfacesin vitro. The attachment defect was related to impaired glucose metabolism, since the noninfectious Mzrlines consumed less glucose, and glucose promoted ATP-independent parasite attachment in the parental lines. Thus, resistance ofGiardiato Mz is accompanied by a glucose metabolism-related attachment defect that can interfere with colonization of the host. Because glucose-metabolizing pathways are important for activation of the prodrug Mz, it follows that a fitness trade-off exists between diminished Mz activation and reduced infectivity, which may explain the observed paucity of clinical Mzrisolates ofGiardia. However, the data also caution that some forms of Mz resistance do not markedly interfere within vivoinfectivity.

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Resistance to the Cyclotide Cycloviolacin O2 in Salmonella enterica Caused by Different Mutations That Often Confer Cross-Resistance or Collateral Sensitivity to Other Antimicrobial Peptides

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00684-17 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 5

Author(s):

Sohaib Z. Malik ◽

Marius Linkevicius ◽

Ulf Göransson ◽

Dan I. Andersson

Keyword(s):

Antimicrobial Peptides ◽

Salmonella Enterica ◽

Cross Resistance ◽

Resistance Mutations ◽

Fitness Costs ◽

Content Type ◽

Collateral Sensitivity ◽

Fitness Advantage ◽

Essential Components

ABSTRACT Antimicrobial peptides (AMPs) are essential components of innate immunity in all living organisms, and these potent broad-spectrum antimicrobials have inspired several antibacterial development programs in the past 2 decades. In this study, the development of resistance to the Gram-negative bacterium-specific peptide cycloviolacin O2 (cyO2), a member of the cyclotide family of plant miniproteins, was characterized in Salmonella enterica serovar Typhimurium LT2. Mutants isolated from serial passaging experiments in increasing concentrations of cyO2 were characterized by whole-genome sequencing. The identified mutations were genetically reconstituted in a wild-type background. The additive effect of mutations was studied in double mutants. Fitness costs, levels of resistance, and cross-resistance to another cyclotide, other peptide and nonpeptide antibiotics, and AMPs were determined. A variety of resistance mutations were identified. Some of these reduced fitness and others had no effect on fitness in vitro, in the absence of cyO2. In mouse competition experiments, four of the cyO2-resistant mutants showed a significant fitness advantage, whereas the effects of the mutations in the others appeared to be neutral. The level of resistance was increased by combining several individual resistance mutations. Several cases of cross-resistance and collateral sensitivity between cyclotides, other AMPs, and antibiotics were identified. These results show that resistance to cyclotides can evolve via several different types of mutations with only minor fitness costs and that these mutations often affect resistance to other AMPs.

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Mutations in cdsA and pgsA Correlate with Daptomycin Resistance in Streptococcus mitis and S. oralis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01531-18 ◽

2018 ◽

Vol 63 (2) ◽

pp. e01531-18 ◽

Cited By ~ 5

Author(s):

Truc T. Tran ◽

Nagendra N. Mishra ◽

Ravin Seepersaud ◽

Lorena Diaz ◽

Rafael Rios ◽

...

Keyword(s):

Cell Membrane ◽

Cell Membranes ◽

Complete Disappearance ◽

Biosynthesis Pathway ◽

Streptococcus Mitis ◽

Content Type ◽

High Level ◽

Viridans Group Streptococci

ABSTRACT We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro. All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.

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Daptomycin Dose-Ranging Evaluation with Single-Dose versus Multidose Ceftriaxone Combinations against Streptococcus mitis/oralis in an Ex Vivo Simulated Endocarditis Vegetation Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00386-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 3

Author(s):

Razieh Kebriaei ◽

Seth A. Rice ◽

Kyle C. Stamper ◽

Ravin Seepersaud ◽

Cristina Garcia-de-la-Maria ◽

...

Keyword(s):

Single Dose ◽

Ex Vivo ◽

Therapeutic Option ◽

Streptococcus Mitis ◽

Oral Flora ◽

Content Type ◽

High Level ◽

Viridans Group Streptococci ◽

Combination Regimens

ABSTRACT The viridans group streptococci (VGS) are a heterogeneous group of organisms which are important components of the normal human oral flora. Among the VGS, the Streptococcus mitis/oralis subgroup is one of the most common causes of infective endocarditis (IE). Daptomycin (DAP) is a potential alternative therapeutic option for invasive S. mitis infections, given high rates of β-lactam resistance and vancomycin tolerance in such strains. However, the ability of these strains to rapidly evolve high-level and durable DAP resistance (DAP-R) is problematic. Recent data suggest that combination DAP-β-lactam therapy circumvents this issue. Human-simulated dose-escalating DAP-alone dose regimens (6, 8, 10, or 12 mg/kg/day times 4 days) versus DAP (6 mg/kg/day) plus ceftriaxone (CRO) (2 g once daily times 4 days or 0.5 g, single dose) were assessed against two prototypical DAP-susceptible (DAP-S) S. mitis/oralis strains (SF100 and 351), as measured by a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs). No DAP-alone regimen was effective, with regrowth of high-level DAP-R isolates observed for both strains over 96-h exposures. Combinations of DAP-CRO with either single- or multidose regimens yielded significant reductions in log10 CFU/g amounts within SEVs for both strains (∼6 log10 CFU/g) within 24 h. In addition, no DAP-R strains were detected in either DAP-CRO combination regimens over the 96-h exposure. In contrast to prior in vitro studies, no perturbations in two key cardiolipin biosynthetic genes (cdsA and pgsA) were identified in DAP-R SEV isolates emerging from strain 351, despite defective phospholipid production. The combination of DAP-CRO warrants further investigation for treatment of IE due to S. mitis/oralis.

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Impact of High-Level Daptomycin Resistance in the Streptococcus mitis Group on Virulence and Survivability during Daptomycin Treatment in Experimental Infective Endocarditis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02418-16 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 3

Author(s):

C. Garcia-de-la-Maria ◽

Y. Q. Xiong ◽

J. M. Pericas ◽

Y. Armero ◽

A. Moreno ◽

...

Keyword(s):

Infective Endocarditis ◽

Parental Strain ◽

Durable Resistance ◽

Relative Fitness ◽

Streptococcus Mitis ◽

Content Type ◽

Target Organs ◽

High Level ◽

Viridans Group Streptococci

ABSTRACT Among the viridans group streptococci, the Streptococcus mitis group is the most common cause of infective endocarditis. These bacteria have a propensity to be β-lactam resistant, as well as to rapidly develop high-level and durable resistance to daptomycin (DAP). We compared a parental, daptomycin-susceptible (DAPs) S. mitis/S. oralis strain and its daptomycin-resistant (DAPr) variant in a model of experimental endocarditis in terms of (i) their relative fitness in multiple target organs in this model (vegetations, kidneys, spleen) when animals were challenged individually and in a coinfection strategy and (ii) their survivability during therapy with daptomycin-gentamicin (an in vitro combination synergistic against the parental strain). The DAPr variant was initially isolated from the cardiac vegetations of animals with experimental endocarditis caused by the parental DAPs strain following treatment with daptomycin. The parental strain and the DAPr variant were comparably virulent when animals were individually challenged. In contrast, in the coinfection model without daptomycin therapy, at both the 106- and 107-CFU/ml challenge inocula, the parental strain outcompeted the DAPr variant in all target organs, especially the kidneys and spleen. When the animals in the coinfection model of endocarditis were treated with DAP-gentamicin, the DAPs strain was completely eliminated, while the DAPr variant persisted in all target tissues. These data underscore that the acquisition of DAPr in S. mitis/S. oralis does come at an intrinsic fitness cost, although this resistance phenotype is completely protective against therapy with a potentially synergistic DAP regimen.

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Pseudomonas aeruginosa High-Level Resistance to Polymyxins and Other Antimicrobial Peptides RequirescprA, a Gene That Is Disrupted in the PAO1 Strain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00904-15 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5377-5387 ◽

Cited By ~ 15

Author(s):

Alina D. Gutu ◽

Nicole S. Rodgers ◽

Jihye Park ◽

Samuel M. Moskowitz

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Peptides ◽

Lipid A ◽

Full Length ◽

Open Reading Frame ◽

Cationic Antimicrobial Peptides ◽

Content Type ◽

Reading Frame ◽

High Level ◽

Pao1 Strain

ABSTRACTThearnlocus, found in many Gram-negative bacterial pathogens, mediates resistance to polymyxins and other cationic antimicrobial peptides through 4-amino-l-arabinose modification of the lipid A moiety of lipopolysaccharide. InPseudomonas aeruginosa, several two-component regulatory systems (TCSs) control thearnlocus, which is necessary but not sufficient for these resistance phenotypes. A previous transposon mutagenesis screen to identify additional polymyxin resistance genes that these systems regulate implicated an open reading frame designated PA1559 in the genome of theP. aeruginosaPAO1 strain. Resequencing of this chromosomal region and bioinformatics analysis for a variety ofP. aeruginosastrains revealed that in the sequenced PAO1 strain, a guanine deletion at the end of PA1559 results in a frameshift and truncation of a full-length open reading frame that also encompasses PA1560 in non-PAO1 strains, such asP. aeruginosaPAK. Deletion analysis in the PAK strain showed that this full-length open reading frame, designatedcprA, is necessary for polymyxin resistance conferred by activating mutations in the PhoPQ, PmrAB, and CprRS TCSs. ThecprAgene was also required for PmrAB-mediated resistance to other cationic antimicrobial peptides in the PAK strain. Repair of the mutatedcprAallele in the PAO1 strain restored polymyxin resistance conferred by an activating TCS mutation. The deletion ofcprAdid not affect thearn-mediated lipid A modification, indicating that the CprA protein is necessary for a different aspect of polymyxin resistance. This protein has a domain structure with a strong similarity to the extended short-chain dehydrogenase/reductase family that comprises isomerases, lyases, and oxidoreductases. These results suggest a new avenue through which to pursue targeted inhibition of polymyxin resistance.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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