Colistin Heteroresistance and Involvement of the PmrAB Regulatory System inAcinetobacter baumannii

ABSTRACTMultidrug-resistantAcinetobacter baumanniiinfection has recently emerged as a worldwide clinical problem, and colistin is increasingly being used as a last-resort therapy. Despite its favorable bacterial killing, resistance and heteroresistance (HR) to colistin have been described. The purpose of the present study was to investigate the role of the PmrAB regulatory pathway in laboratory-selected mutants representative of global epidemic strains. From three unrelatedA. baumanniiclinical strains (sequence types 2, 3, and 20), eight colistin-resistant mutants were selected. Half of the mutants showed HR to colistin according to the reference method (population analysis profiling), whereas the other half exhibited stable resistance. M12I mutation withinpmrAand M308R, S144KLAGS, and P170L mutations forpmrBwere associated with HR to colistin, while T235I, A226T, and P233S mutations withinpmrBwere associated with stable resistance. The transcript levels of thepmrCABoperon were upregulated in all the mutants. Compensatory mutations were explored for some mutants. A single mutant (T235I mutant) displayed a compensatory mutation through ISAba1mobilization within thepmrBgene that was associated with the loss of colistin resistance. The mutant resistance phenotype associated with T235I was partially restored in atrans-complementation assay turning to HR. The level of colistin resistance was correlated with the level of expression ofpmrCin thetrans-complemented strains. This report shows the role of different mutations in the PmrAB regulatory pathway and warns of the development of colistin HR that could be present but not easily detected through routine testing.

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Colistin heteroresistance and the involvement of the PmrAB regulatory system in Acinetobacter baumannii

10.1101/307132 ◽

2018 ◽

Author(s):

Yannick Charretier ◽

Seydina M. Diene ◽

Damien Baud ◽

Sonia Chatellier ◽

Emmanuelle Santiago-Allexant ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Population Analysis ◽

Regulatory System ◽

Clinical Problem ◽

Multidrug Resistant ◽

Regulatory Pathway ◽

Colistin Resistance ◽

Bacterial Killing ◽

One Step

AbstractMultidrug-resistant Acinetobacter baumannii infection has recently emerged as a worldwide clinical problem and colistin is increasingly being used as last resort therapy. Despite its favorable bacterial killing, resistance and heteroresistance to colistin have been described. Mutations in the PmrAB regulatory pathway have been already associated with colistin resistance whereas the mechanisms for heteroresistance remain largely unknown. The purpose of the present study is to investigate the role of PmrAB in laboratory-selected mutants representative of global epidemic strains. During brief colistin exposure, colistin resistant and colistin heteroresistant mutants were selected in a one-step strategy. Population Analysis Profiling (PAP) was performed to confirm the suspected phenotype. Upon withdrawal of selective pressure, compensatory mutations were evaluated in another one-step strategy. A trans-complementation assay was designed to delineate the involvement of the PmrAB regulatory system using qPCR and PAP. Mutations in the PmrAB regulatory pathway were associated with colistin resistance and colistin heteroresistance as well. The transcomplementation assay provides a proof for the role played by changes in the PmrAB regulatory pathway. The level of colistin resistance is correlated to the level of expression of pmrC. The resistance phenotype was partially restored since the complemented strain became heteroresistant. This report shows the role of different mutations in the PmrAB regulatory pathway and warns on the development of colistin heteroresistance that could be present but not easily detected with routine testing.

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Synergistic Activity of Colistin and Rifampin Combination against Multidrug-Resistant Acinetobacter baumannii in anIn VitroPharmacokinetic/Pharmacodynamic Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00703-13 ◽

2013 ◽

Vol 57 (8) ◽

pp. 3738-3745 ◽

Cited By ~ 60

Author(s):

Hee Ji Lee ◽

Phillip J. Bergen ◽

Jurgen B. Bulitta ◽

Brian Tsuji ◽

Alan Forrest ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Population Analysis ◽

Multidrug Resistant ◽

Synergistic Activity ◽

Colistin Resistance ◽

Bacterial Killing ◽

Content Type ◽

High Inoculum ◽

Combination Regimens

ABSTRACTCombination therapy may be required for multidrug-resistant (MDR)Acinetobacter baumannii. This study systematically investigated bacterial killing and emergence of colistin resistance with colistin and rifampin combinations against MDRA. baumannii. Studies were conducted over 72 h in anin vitropharmacokinetic (PK)/pharmacodynamic (PD) model at inocula of ∼106and ∼108CFU/ml using two MDR clinical isolates ofA. baumannii, FADDI-AB030 (colistin susceptible) and FADDI-AB156 (colistin resistant). Three combination regimens achieving clinically relevant concentrations (constant colistin concentration of 0.5, 2, or 5 mg/liter and a rifampin maximum concentration [Cmax] of 5 mg/liter every 24 hours; half-life, 3 h) were investigated. Microbiological response was measured by serial bacterial counts. Population analysis profiles assessed emergence of colistin resistance. Against both isolates, combinations resulted in substantially greater killing at the low inoculum; combinations containing 2 and 5 mg/liter colistin increased killing at the high inoculum. Combinations were additive or synergistic at 6, 24, 48, and 72 h with all colistin concentrations against FADDI-AB030 and FADDI-AB156 in, respectively, 8 and 11 of 12 cases (i.e., all 3 combinations) at the 106-CFU/ml inoculum and 8 and 7 of 8 cases with the 2- and 5-mg/liter colistin regimens at the 108-CFU/ml inoculum. For FADDI-AB156, killing by the combination was ∼2.5 to 7.5 and ∼2.5 to 5 log10CFU/ml greater at the low inoculum (all colistin concentrations) and high inoculum (2 and 5 mg/liter colistin), respectively. Emergence of colistin-resistant subpopulations was completely suppressed in the colistin-susceptible isolate with all combinations at both inocula. Our study provides important information for optimizing colistin-rifampin combinations against colistin-susceptible and -resistant MDRA. baumannii.

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The Combination of Colistin and Doripenem Is Synergistic against Klebsiella pneumoniae at Multiple Inocula and Suppresses Colistin Resistance in anIn VitroPharmacokinetic/Pharmacodynamic Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01064-12 ◽

2012 ◽

Vol 56 (10) ◽

pp. 5103-5112 ◽

Cited By ~ 63

Author(s):

Zakuan Z. Deris ◽

Heidi H. Yu ◽

Kathryn Davis ◽

Rachel L. Soon ◽

Jovan Jacob ◽

...

Keyword(s):

Combination Therapy ◽

Klebsiella Pneumoniae ◽

Population Analysis ◽

Multidrug Resistant ◽

Pharmacodynamic Model ◽

Resistant Isolate ◽

Colistin Resistance ◽

Bacterial Killing ◽

Content Type

ABSTRACTMultidrug-resistant (MDR)Klebsiella pneumoniaemay require combination therapy. We systematically investigated bacterial killing with colistin and doripenem mono- and combination therapy against MDRK. pneumoniaeand emergence of colistin resistance. A one-compartmentin vitropharmacokinetic/pharmacodynamic model was employed over a 72-h period with two inocula (∼106and ∼108CFU/ml); a colistin-heteroresistant reference strain (ATCC 13883) and three clinical isolates (colistin-susceptible FADDI-KP032 [doripenem resistant], colistin-heteroresistant FADDI-KP033, and colistin-resistant FADDI-KP035) were included. Four combinations utilizing clinically achievable concentrations were investigated. Microbiological responses were examined by determining log changes and population analysis profiles (for emergence of colistin resistance) over 72 h. Against colistin-susceptible and -heteroresistant isolates, combinations of colistin (constant concentration regimens of 0.5 or 2 mg/liter) plus doripenem (steady-state peak concentration [Cmax] of 2.5 or 25 mg/liter over 8 h; half-life, 1.5 h) generally resulted in substantial improvements in bacterial killing at both inocula. Combinations were additive or synergistic against ATCC 13883, FADDI-KP032, and FADDI-KP033 in 9, 9, and 14 of 16 cases (4 combinations at 6, 24, 48, and 72 h) at the 106-CFU/ml inoculum and 14, 11, and 12 of 16 cases at the 108-CFU/ml inoculum, respectively. Combinations at the highest dosage regimens resulted in undetectable bacterial counts at 72 h in 5 of 8 cases (4 isolates at 2 inocula). Emergence of colistin-resistant subpopulations in colistin-susceptible and -heteroresistant isolates was virtually eliminated with combination therapy. Against the colistin-resistant isolate, colistin at 2 mg/liter plus doripenem (Cmax, 25 mg/liter) at the low inoculum improved bacterial killing. This investigation provides important information for optimization of colistin-doripenem combinations.

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Resistance and Heteroresistance to Colistin in Pseudomonas aeruginosa Isolates from Wenzhou, China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00556-19 ◽

2019 ◽

Vol 63 (10) ◽

Cited By ~ 8

Author(s):

Jie Lin ◽

Chunquan Xu ◽

Renchi Fang ◽

Jianming Cao ◽

Xiucai Zhang ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

Population Analysis ◽

Colistin Resistance ◽

Content Type ◽

Maldi Tof Ms ◽

Expression Levels ◽

Maldi Tof ◽

Broth Microdilution Method ◽

Tof Ms

ABSTRACT The goal was to investigate the mechanisms of colistin resistance and heteroresistance in Pseudomonas aeruginosa clinical isolates. Colistin resistance was determined by the broth microdilution method. Colistin heteroresistance was evaluated by population analysis profiling. Time-kill assays were also conducted. PCR sequencing was performed to detect the resistance genes among (hetero)resistant isolates, and quantitative real-time PCR assays were performed to determine their expression levels. Pulsed-field gel electrophoresis and multilocus sequence typing were performed. Lipid A characteristics were determined via matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS). Two resistant isolates and 9 heteroresistant isolates were selected in this study. Substitutions in PmrB were detected in 2 resistant isolates. Among heteroresistant isolates, 8 of 9 heteroresistant isolates had nonsynonymous PmrB substitutions, and 2 isolates, including 1 with a PmrB substitution, had PhoQ alterations. Correspondingly, the expression levels of pmrA or phoP were upregulated in PmrB- or PhoQ-substituted isolates. One isolate also found alterations in ParRS and CprRS. The transcript levels of the pmrH gene were observed to increase across all investigated isolates. MALDI-TOF MS showed additional 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties in lipid A profiles in (hetero)resistant isolates. In conclusion, both colistin resistance and heteroresistance in P. aeruginosa in this study mainly involved alterations of the PmrAB regulatory system. There were strong associations between mutations in specific genetic loci for lipid A synthesis and regulation of modifications to lipid A. The transition of colistin heteroresistance to resistance should be addressed in future clinical surveillance.

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Spontaneous Gac Mutants of Pseudomonas Biological Control Strains: Cheaters or Mutualists?

Applied and Environmental Microbiology ◽

10.1128/aem.00679-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7227-7235 ◽

Cited By ~ 31

Author(s):

William W. Driscoll ◽

John W. Pepper ◽

Leland S. Pierson ◽

Elizabeth A. Pierson

Keyword(s):

Alternative Hypothesis ◽

Natural Populations ◽

Regulatory System ◽

Wild Type ◽

Content Type ◽

The Public ◽

Extracellular Metabolites ◽

Mutualistic Interaction ◽

Alternative Hypotheses

ABSTRACTBacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are “cheaters” that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacteriumPseudomonas chlororaphisstrain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions.

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The role of Akkermansia muciniphila in obesity, diabetes and atherosclerosis

Journal of Medical Microbiology ◽

10.1099/jmm.0.001435 ◽

2021 ◽

Vol 70 (10) ◽

Author(s):

Alka Hasani ◽

Saba Ebrahimzadeh ◽

Fatemeh Hemmati ◽

Aytak Khabbaz ◽

Akbar Hasani ◽

...

Keyword(s):

Type Species ◽

Anaerobic Bacteria ◽

Regulatory System ◽

Content Type ◽

Akkermansia Muciniphila ◽

Link Type ◽

Animal Trials ◽

Obesity And Diabetes ◽

Underlying Mechanisms

Alteration in the composition of the gut microbiota can lead to a number of chronic clinical diseases. Akkermansia muciniphila is an anaerobic bacteria constituting 3–5% of the gut microbial community in healthy adults. This bacterium is responsible for degenerating mucin in the gut; its scarcity leads to diverse clinical disorders. In this review, we focus on the role of A. muciniphila in diabetes, obesity and atherosclerosis, as well as the use of this bacterium as a next-generation probiotic. In regard to obesity and diabetes, human and animal trials have shown that A. muciniphila controls the essential regulatory system of glucose and energy metabolism. However, the underlying mechanisms by which A. muciniphila alleviates the complications of obesity, diabetes and atherosclerosis are unclear. At the same time, its abundance suggests improved metabolic disorders, such as metabolic endotoxemia, adiposity insulin resistance and glucose tolerance. The role of A. muciniphila is implicated in declining aortic lesions and atherosclerosis. Well-characterized virulence factors, antigens and cell wall extracts of A. muciniphila may act as effector molecules in these diseases. These molecules may provide novel mechanisms and strategies by which this bacterium could be used as a probiotic for the treatment of obesity, diabetes and atherosclerosis.

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CsrA (BB0184) Is Not Involved in Activation of the RpoN-RpoS Regulatory Pathway in Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.01555-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1511-1522 ◽

Cited By ~ 10

Author(s):

Zhiming Ouyang ◽

Jianli Zhou ◽

Michael V. Norgard

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

Animal Studies ◽

Expression System ◽

Regulatory Pathway ◽

Content Type ◽

Mouse Tissues ◽

Differential Gene ◽

Carbon Storage Regulator

ABSTRACTBorrelia burgdorferiencodes a homologue of the bacterial carbon storage regulator A (CsrA). Recently, it was reported that CsrA contributes toB. burgdorferiinfectivity and is required for the activation of the central RpoN-RpoS regulatory pathway. However, many questions concerning the function of CsrA inB. burgdorferigene regulation remain unanswered. In particular, there are conflicting reports concerning the molecular details of how CsrA may modulaterpoSexpression and, thus, how CsrA may influence the RpoN-RpoS pathway inB. burgdorferi. To address these key discrepancies, we examined the role of CsrA in differential gene expression in the Lyme disease spirochete. Upon engineering an induciblecsrAexpression system inB. burgdorferi, controlled hyperexpression of CsrA in a merodiploid strain did not significantly alter the protein and transcript levels ofbosR,rpoS, and RpoS-dependent genes (such asospCanddbpA). In addition, we constructed isogeniccsrAmutants in two widely used infectiousB. burgdorferistrains. When expression ofbosR,rpoS,ospC, anddbpAwas compared between thecsrAmutants and their wild-type counterparts, no detectable differences were observed. Finally, animal studies indicated that thecsrAmutants remained infectious for and virulent in mice. Analyses ofB. burgdorferigene expression in mouse tissues showed comparable levels ofrpoStranscripts by thecsrAmutants and the parental strains. Taken together, these results constitute compelling evidence that CsrA is not involved in activation of the RpoN-RpoS pathway and is dispensable for mammalian infectious processes carried out byB. burgdorferi.

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A Low-Affinity Penicillin-Binding Protein 2x Variant Is Required for Heteroresistance in Streptococcus pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02547-14 ◽

2014 ◽

Vol 58 (7) ◽

pp. 3934-3941 ◽

Cited By ~ 14

Author(s):

Hansjürg Engel ◽

Moana Mika ◽

Dalia Denapaite ◽

Regine Hakenbeck ◽

Kathrin Mühlemann ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Abc Transporter ◽

Binding Protein ◽

Population Analysis ◽

Resistance Testing ◽

Penicillin Binding Protein ◽

Secondary Resistance ◽

Content Type ◽

Abc Transporter Genes

ABSTRACTHeteroresistance to penicillin inStreptococcus pneumoniaeis the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain23F2349in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found thatpbp2x, but notpbp2borpbp1aalone, conferred heteroresistance to R6. However, a change ofpbp2xexpression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent β-lactam resistance, was assessed by PAP onciaRdisruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinitypbp2xundergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded bypstS,phoU,pstB, andpstCin these highly resistant subpopulations. In conclusion, the presence of low-affinitypbp2xenables certain pneumococcal colonies to survive in the presence of β-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.

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Magnesium Sensing Regulates Intestinal Colonization of Enterohemorrhagic Escherichia coli O157:H7

mBio ◽

10.1128/mbio.02470-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Yutao Liu ◽

Runhua Han ◽

Junyue Wang ◽

Pan Yang ◽

Fang Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Large Intestine ◽

Intestinal Tract ◽

Regulatory System ◽

Regulatory Pathway ◽

Content Type ◽

E Coli ◽

Low Magnesium ◽

Enterocyte Effacement

ABSTRACT The large intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 detects host cues to regulate virulence gene expression during colonization and infection. However, virulence regulatory mechanisms of EHEC O157:H7 in the human large intestine are not fully understood. Herein, we identified a virulence-regulating pathway where the PhoQ/PhoP two-component regulatory system senses low magnesium levels and signals to the O island 119-encoded Z4267 (LmiA; low magnesium-induced regulator A), directly activating loci of enterocyte effacement genes to promote EHEC O157:H7 adherence in the large intestine. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, feeding mice a magnesium-rich diet significantly reduced EHEC O157:H7 adherence in vivo. This LmiA-mediated virulence regulatory pathway is also conserved among several EHEC and enteropathogenic E. coli serotypes; therefore, our findings support the use of magnesium as a dietary supplement and provide greater insights into the dietary cues that can prevent enteric infections. IMPORTANCE Sensing specific gut metabolites is an important strategy for inducing crucial virulence programs by enterohemorrhagic Escherichia coli (EHEC) O157:H7 during colonization and infection. Here, we identified a virulence-regulating pathway wherein the PhoQ/PhoP two-component regulatory system signals to the O island 119-encoded low magnesium-induced regulator A (LmiA), which, in turn, activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence in the low-magnesium conditions of the large intestine. This regulatory pathway is widely present in a range of EHEC and enteropathogenic E. coli (EPEC) serotypes. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, mice fed a magnesium-rich diet showed significantly reduced EHEC O157:H7 adherence in vivo, indicating that magnesium may help in preventing EHEC and EPEC infection in humans.

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The Mga Regulon but Not Deoxyribonuclease Sda1 of Invasive M1T1 Group A Streptococcus Contributes toIn VivoSelection of CovRS Mutations and Resistance to Innate Immune Killing Mechanisms

Infection and Immunity ◽

10.1128/iai.00857-15 ◽

2015 ◽

Vol 83 (11) ◽

pp. 4293-4303 ◽

Cited By ~ 9

Author(s):

Guanghui Liu ◽

Wenchao Feng ◽

Dengfeng Li ◽

Mengyao Liu ◽

Daniel C. Nelson ◽

...

Keyword(s):

Group A Streptococcus ◽

Regulatory System ◽

Innate Immune ◽

M Protein ◽

Content Type ◽

Group A ◽

Innate Immune Evasion ◽

Selection Of

ABSTRACTInvasive M1T1 group AStreptococcus(GAS) can have a mutation in the regulatory system CovRS, and this mutation can render strains hypervirulent. Interestingly, via mechanisms that are not well understood, the host innate immune system's neutrophils select spontaneous M1T1 GAS CovRS hypervirulent mutants, thereby enhancing the pathogen's ability to evade immune killing. It has been reported that the DNase Sda1 is critical for the resistance of M1T1 strain 5448 to killing in human blood and provides pressure forin vivoselection of CovRS mutations. We reexamined the role of Sda1 in the selection of CovRS mutations and in GAS innate immune evasion. Deletion ofsda1or all DNase genes in M1T1 strain MGAS2221 did not alter emergence of CovRS mutants during murine infection. Deletion ofsda1in strain 5448 resulted in Δsda1mutants with (5448 Δsda1M+strain) and without (5448 Δsda1M−strain) M protein production. The 5448 Δsda1M+strain accumulated CovRS mutationsin vivoand resisted killing in the bloodstream, whereas the 5448 Δsda1M−strain lostin vivoselection of CovRS mutations and was sensitive to killing. The deletion ofemmand a spontaneous Mga mutation in MGAS2221 reduced and preventedin vivoselection for CovRS mutants, respectively. Thus, in contrast to previous reports, Sda1 is not critical forin vivoselection of invasive M1T1 CovRS mutants and GAS resistance to innate immune killing mechanisms. In contrast, M protein and other Mga-regulated proteins contribute to thein vivoselection of M1T1 GAS CovRS mutants. These findings advance the understanding of the progression of invasive M1T1 GAS infections.

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