Cellular Growth Arrest and Efflux Pumps Are Associated With Antibiotic Persisters in Streptococcus pyogenes Induced in Biofilm-Like Environments

Streptococcus pyogenes (group A Streptococcus-GAS) is an important pathogen for humans. GAS has been associated with severe and invasive diseases. Despite the fact that these bacteria remain universally susceptible to penicillin, therapeutic failures have been reported in some GAS infections. Many hypotheses have been proposed to explain these antibiotic-unresponsive infections; however, none of them have fully elucidated this phenomenon. In this study, we show that GAS strains have the ability to form antimicrobial persisters when inoculated on abiotic surfaces to form a film of bacterial agglomerates (biofilm-like environment). Our data suggest that efflux pumps were possibly involved in this phenomenon. In fact, gene expression assays by real-time qRT-PCR showed upregulation of some genes associated with efflux pumps in persisters arising in the presence of penicillin. Phenotypic reversion assay and whole-genome sequencing indicated that this event was due to non-inherited resistance mechanisms. The persister cells showed downregulation of genes associated with protein biosynthesis and cell growth, as demonstrated by gene expression assays. Moreover, the proteomic analysis revealed that susceptible cells express higher levels of ribosome proteins. It is remarkable that previous studies have reported the recovery of S. pyogenes viable cells from tissue biopsies of patients presented with GAS invasive infections and submitted to therapy with antibiotics. The persistence phenomenon described herein brings new insights into the origin of therapeutic failures in S. pyogenes infections. Multifactorial mechanisms involving protein synthesis inhibition, cell growth impairment and efflux pumps seem to play roles in the formation of antimicrobial persisters in S. pyogenes.

Download Full-text

Occurrence of antimicrobial persistence in Streptococcus pyogenes

10.21203/rs.3.rs-129287/v2 ◽

2021 ◽

Author(s):

Caroline L. Martini ◽

Amada Z. Coronado ◽

Maria Celeste N. Melo ◽

Clarice N. Gobbi ◽

Úrsula S. Lopez ◽

...

Keyword(s):

Gene Expression ◽

Cell Growth ◽

Streptococcus Pyogenes ◽

Efflux Pump ◽

Protein Biosynthesis ◽

Resistance Mechanisms ◽

Whole Genome Sequence ◽

Abiotic Surfaces ◽

Invasive Infections ◽

Group A

Abstract Streptococcus pyogenes (group A Streptococcus-GAS) is an important pathogen for humans. GAS has been associated with severe and invasive diseases. Despite the fact that these bacteria remain universally susceptible to penicillin, therapeutic failures have been reported in some GAS infections. Many hypotheses have been proposed to explain these antibiotic-unresponsive infections, however none of them have fully elucidated this phenomenon. In this study, we show that GAS strains have the ability to form antimicrobial-persistent cells when growing on abiotic surfaces in a biofilm-like environment. Our data suggest that efflux activity was possibly involved in this phenomenon. In fact, gene expression assays by real-time qRT-PCR showed upregulation of some genes associated with efflux pumps in persistent cells arising in the presence of penicillin. Phenotypic reversion assay and whole-genome sequence analysis indicated that this event was due to non-inherited resistance mechanisms. The small persistent colonies showed downregulation of genes associated with protein biosynthesis and cell growth, as demonstrated by gene expression assays. Moreover, the proteomic analysis showed that susceptible cells express higher levels of ribosome proteins. It is conceivable to suppose that the generation of persistent cells by S. pyogenes might a mechanism of antimicrobial refractory during the course of real GAS invasive infections, which has been overlooked. Accordingly, the phenomenon described here might shed some light on the origin of therapeutic failures in S. pyogenes infections. Multifactorial mechanisms involving efflux pump activity, protein synthesis inhibition and cell-growth impairment seem to play roles in generation of GAS-persistent cells.

Download Full-text

High Cell Density and Antimicrobial Persistence in Streptococcus Pyogenes

10.21203/rs.3.rs-129287/v1 ◽

2020 ◽

Author(s):

Caroline Martini ◽

Amada Coronado ◽

Maria Celeste Melo ◽

Clarice Gobbi ◽

Úrsula Lopez ◽

...

Keyword(s):

Gene Expression ◽

Cell Density ◽

Streptococcus Pyogenes ◽

High Cell Density ◽

Protein Biosynthesis ◽

Bacterial Load ◽

Resistance Mechanisms ◽

High Cell ◽

Clinical Resistance ◽

Invasive Infections

Abstract Streptococcus pyogenes (group A Streptococcus-GAS) is an important pathogen for humans. GAS has been associated with severe and invasive diseases. Despite the fact that these bacteria remain universally susceptible to penicillin, therapeutic failures have been reported in some GAS infections. Many hypotheses have been proposed to explain these antibiotic-unresponsive infections, however none of them has fully elucidated this phenomenon. In this study, antimicrobial persistence emerged when GAS strains were grown at high cell density. Strong efflux activity was detected, and gene expression assays by real-time qRT-PCR showed upregulation of some genes associated with efflux pumps in persistent cells arising in the presence of penicillin. Subsequent phenotypic reversion and whole-genome sequencing indicated that this event was due to non-inherited resistance mechanisms. The tiny persistent colonies showed downregulation of genes associated with protein biosynthesis and cell growth, as demonstrated by gene expression assays. Moreover, proteomic analyses showed that susceptible cells express higher levels of ribosome proteins. The generation of persistent cells due to high bacterial load might be an important mechanism of clinical resistance in GAS invasive infections that has been overlooked. The phenomenon described here might shed some light on the origin of therapeutic failures in S. pyogenes infections.

Download Full-text

Distribution of emm type and antibiotic susceptibility of group A streptococci causing invasive and noninvasive disease

Journal of Medical Microbiology ◽

10.1099/jmm.0.2008/002642-0 ◽

2008 ◽

Vol 57 (11) ◽

pp. 1383-1388 ◽

Cited By ~ 43

Author(s):

Takeaki Wajima ◽

Somay Y. Murayama ◽

Katsuhiko Sunaoshi ◽

Eiichi Nakayama ◽

Keisuke Sunakawa ◽

...

Keyword(s):

Streptococcus Pyogenes ◽

Macrolide Antibiotic ◽

Group A Streptococcus ◽

Acute Otitis Media ◽

Toxic Shock ◽

Medical Institutions ◽

Invasive Infections ◽

Group A ◽

Resistant Strains ◽

Emm Type

To determine the prevalence of macrolide antibiotic and levofloxacin resistance in infections with Streptococcus pyogenes (group A streptococcus or GAS), strains were collected from 45 medical institutions in various parts of Japan between October 2003 and September 2006. Four hundred and eighty-two strains from patients with GAS infections were characterized genetically. Strains were classified into four groups according to the type of infection: invasive infections (n=74) including sepsis, cellulitis and toxic-shock-like syndrome; acute otitis media (AOM; n=23); abscess (n=53); and pharyngotonsillitis (n=332). Among all strains, 32 emm types were identified; emm1 was significantly more common in invasive infections (39.2 %) and AOM (43.5 %) than in abscesses (3.8 %) or pharyngotonsillitis (10.2 %). emm12 and emm4 each accounted for 23.5 % of pharyngotonsillitis cases. Susceptibility of GAS strains to eight β-lactam agents was excellent, with MICs of 0.0005–0.063 μg ml−1. Macrolide-resistant strains accounted for 16.2 % of all strains, while the percentages of strains possessing the resistance genes erm(A), erm(B) and mef(A) were 2.5 %, 6.2 % and 7.5 %, respectively. Although no strains with high resistance to levofloxacin were found, strains with an MIC of 2–4 μg ml−1 (17.4 %) had amino acid substitutions at either Ser-79 or Asp-83 in ParC. These levofloxacin-intermediately resistant strains included 16 emm types, but macrolide-resistant strains were more likely than others to represent certain emm types.

Download Full-text

Streptococcus pyogenes evades adaptive immunity through specific IgG glycan hydrolysis

Journal of Experimental Medicine ◽

10.1084/jem.20190293 ◽

2019 ◽

Vol 216 (7) ◽

pp. 1615-1629 ◽

Cited By ~ 2

Author(s):

Andreas Naegeli ◽

Eleni Bratanis ◽

Christofer Karlsson ◽

Oonagh Shannon ◽

Raja Kalluru ◽

...

Keyword(s):

Streptococcus Pyogenes ◽

Vaccine Development ◽

Group A Streptococcus ◽

Invasive Infection ◽

Invasive Infections ◽

Life Threatening ◽

Group A ◽

Specific Igg

Streptococcus pyogenes (Group A streptococcus; GAS) is a human pathogen causing diseases from uncomplicated tonsillitis to life-threatening invasive infections. GAS secretes EndoS, an endoglycosidase that specifically cleaves the conserved N-glycan on IgG antibodies. In vitro, removal of this glycan impairs IgG effector functions, but its relevance to GAS infection in vivo is unclear. Using targeted mass spectrometry, we characterized the effects of EndoS on host IgG glycosylation during the course of infections in humans. Substantial IgG glycan hydrolysis occurred at the site of infection and systemically in the severe cases. We demonstrated decreased resistance to phagocytic killing of GAS lacking EndoS in vitro and decreased virulence in a mouse model of invasive infection. This is the first described example of specific bacterial IgG glycan hydrolysis during infection and thereby verifies the hypothesis that EndoS modifies antibodies in vivo. This mechanisms of immune evasion could have implications for treatment of severe GAS infections and for future efforts at vaccine development.

Download Full-text

Unraveling the Regulatory Network in Streptococcus pyogenes: the Global Response Regulator CovR Represses rivR Directly

Journal of Bacteriology ◽

10.1128/jb.01026-06 ◽

2006 ◽

Vol 189 (4) ◽

pp. 1459-1463 ◽

Cited By ~ 22

Author(s):

Samantha A. Roberts ◽

Gordon G. Churchward ◽

June R. Scott

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Streptococcus Pyogenes ◽

Regulatory Network ◽

Group A Streptococcus ◽

Response Regulator ◽

Master Regulator ◽

Global Response ◽

Conserved Sequences ◽

Group A

ABSTRACT The response regulator CovR acts as a master regulator of virulence in Streptococcus pyogenes by repressing transcription of approximately 15% of the group A streptococcus genome directly or indirectly. We demonstrate that phosphorylated CovR represses transcription of rivR directly by binding to conserved sequences located downstream from the promoter to block procession of RNA polymerase. This establishes the first link in a regulatory network where CovR interacts directly with other proteins that modulate gene expression.

Download Full-text

Invasive infections due to Streptococcus pyogenes: seasonal variation of severity and clinical characteristics, Iceland, 1975 to 2012

Eurosurveillance ◽

10.2807/1560-7917.es2014.19.17.20784 ◽

2014 ◽

Vol 19 (17) ◽

Cited By ~ 23

Author(s):

L B Olafsdottir ◽

H Erlendsdóttir ◽

J Melo-Cristino ◽

D M Weinberger ◽

M Ramirez ◽

...

Keyword(s):

Streptococcus Pyogenes ◽

Clinical Characteristics ◽

Population Based ◽

Host Susceptibility ◽

Population Based Study ◽

Virulent Strains ◽

Invasive Infections ◽

Group A ◽

Severe Infections

Epidemiology and clinical characteristics of invasive Group A streptococcal infections (IGASI) are highly variable. Long-term studies are needed to understand the interplay between epidemiology and virulence. In a population-based study of IGASI in Iceland from 1975 to 2012, 288 cases were identified by positive cultures from normally sterile body sites. Charts were reviewed retrospectively and emm-types of viable Streptococcus pyogenes isolates (n=226) determined. Comparing the first and last decade of the study period, IGASI incidence increased from 1.09 to 3.96 cases per 100,000 inhabitants per year. The most common were emm types 1 (25%), 28 (11%) and 89 (11%); emm1 strains were most likely to cause severe infections. Infections in adults were significantly more likely to be severe during the seasonal peak from January to April (risk ratio: 2.36, 95% confidence interval: 1.34–4.15). Significant seasonal variability in severity was noted among patients with diagnosis of sepsis, respiratory infection and cellulitis, with 38% of severe infections in January to April compared with 16% in other months (p<0.01). A seasonal increase in severity of IGASI suggested that generalised seasonal increase in host susceptibility, rather than introduction of more virulent strains may play a role in the pathogenesis of these potentially fatal infections.

Download Full-text

CovS Simultaneously Activates and Inhibits the CovR-Mediated Repression of Distinct Subsets of Group A Streptococcus Virulence Factor-Encoding Genes

Infection and Immunity ◽

10.1128/iai.01560-08 ◽

2009 ◽

Vol 77 (8) ◽

pp. 3141-3149 ◽

Cited By ~ 83

Author(s):

Jeanette Treviño ◽

Nataly Perez ◽

Esmeralda Ramirez-Peña ◽

Zhuyun Liu ◽

Samuel A. Shelburne ◽

...

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Regulatory Function ◽

Upper Respiratory Tract ◽

Wild Type ◽

In Vivo Models ◽

Mutant Strains ◽

Invasive Infections ◽

Group A

ABSTRACT To colonize and cause disease at distinct anatomical sites, bacterial pathogens must tailor gene expression in a microenvironment-specific manner. The molecular mechanisms that control the ability of the human bacterial pathogen group A Streptococcus (GAS) to transition between infection sites have yet to be fully elucidated. A key regulator of GAS virulence gene expression is the CovR-CovS two-component regulatory system (also known as CsrR-CsrS). covR and covS mutant strains arise spontaneously during invasive infections and, in in vivo models of infection, rapidly become dominant. Here, we compared wild-type GAS with covR, covS, and covRS isogenic mutant strains to investigate the heterogeneity in the types of natural mutations that occur in covR and covS and the phenotypic consequences of covR or covS mutation. We found that the response regulator CovR retains some regulatory function in the absence of CovS and that CovS modulates CovR to significantly enhance repression of one group of genes (e.g., the speA, hasA, and ska genes) while it reduces repression of a second group of genes (e.g., the speB, grab, and spd3 genes). We also found that different in vivo-induced covR mutations can lead to strikingly different transcriptomes. While covS mutant strains show increased virulence in several invasive models of infection, we determined that these mutants are significantly outcompeted by wild-type GAS during growth in human saliva, an ex vivo model of upper respiratory tract infection. We propose that CovS-mediated regulation of CovR activity plays an important role in the ability of GAS to cycle between pharyngeal and invasive infections.

Download Full-text

Streptolysin O and NAD-Glycohydrolase Prevent Phagolysosome Acidification and Promote Group A Streptococcus Survival in Macrophages

mBio ◽

10.1128/mbio.01690-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 47

Author(s):

Benedicte Bastiat-Sempe ◽

John F. Love ◽

Natalie Lomayesva ◽

Michael R. Wessels

Keyword(s):

Streptococcus Pyogenes ◽

Intracellular Survival ◽

Streptococcal Toxic Shock Syndrome ◽

Simple Diffusion ◽

Content Type ◽

Nad Glycohydrolase ◽

Macrophage Phagocytosis ◽

Streptolysin O ◽

Invasive Infections ◽

Group A

ABSTRACTGroup AStreptococcus(GAS,Streptococcus pyogenes) is an ongoing threat to human health as the agent of streptococcal pharyngitis, skin and soft tissue infections, and life-threatening conditions such as necrotizing fasciitis and streptococcal toxic shock syndrome. In animal models of infection, macrophages have been shown to contribute to host defense against GAS infection. However, as GAS can resist killing by macrophagesin vitroand induce macrophage cell death, it has been suggested that GAS intracellular survival in macrophages may enable persistent infection. Using isogenic mutants, we now show that the GAS pore-forming toxin streptolysin O (SLO) and its cotoxin NAD-glycohydrolase (NADase) mediate GAS intracellular survival and cytotoxicity for macrophages. Unexpectedly, the two toxins did not inhibit fusion of GAS-containing phagosomes with lysosomes but rather prevented phagolysosome acidification. SLO served two essential functions, poration of the phagolysosomal membrane and translocation of NADase into the macrophage cytosol, both of which were necessary for maximal GAS intracellular survival. Whereas NADase delivery to epithelial cells is mediated by SLO secreted from GAS bound to the cell surface, in macrophages, the source of SLO and NADase is GAS contained within phagolysosomes. We found that transfer of NADase from the phagolysosome to the macrophage cytosol occurs not by simple diffusion through SLO pores but rather by a specific translocation mechanism that requires the N-terminal translocation domain of NADase. These results illuminate the mechanisms through which SLO and NADase enable GAS to defeat macrophage-mediated killing and provide new insight into the virulence of a major human pathogen.IMPORTANCEMacrophages constitute an important element of the innate immune response to mucosal pathogens. They ingest and kill microbes by phagocytosis and secrete inflammatory cytokines to recruit and activate other effector cells. Group AStreptococcus(GAS,Streptococcus pyogenes), an important cause of pharyngitis and invasive infections, has been shown to resist killing by macrophages. We find that GAS resistance to macrophage killing depends on the GAS pore-forming toxin streptolysin O (SLO) and its cotoxin NAD-glycohydrolase (NADase). GAS bacteria are internalized by macrophage phagocytosis but resist killing by secreting SLO, which damages the phagolysosome membrane, prevents phagolysosome acidification, and translocates NADase from the phagolysosome into the macrophage cytosol. NADase augments SLO-mediated cytotoxicity by depleting cellular energy stores. These findings may explain the nearly universal production of SLO by GAS clinical isolates and the association of NADase with the global spread of a GAS clone implicated in invasive infections.

Download Full-text

Changes in emm types and superantigen gene content of Streptococcus pyogenes causing invasive infections in Portugal

Scientific Reports ◽

10.1038/s41598-019-54409-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

A. Friães ◽

◽

J. Melo-Cristino ◽

M. Ramirez

Keyword(s):

Streptococcus Pyogenes ◽

Susceptibility Testing ◽

Gene Profiling ◽

Short Term ◽

Genomic Variants ◽

Increasing Trend ◽

Invasive Infections ◽

Group A ◽

Profile Changes ◽

Clonal Composition

AbstractFluctuations in the clonal composition of Group A Streptococcus (GAS) have been associated with the emergence of successful lineages and with upsurges of invasive infections (iGAS). This study aimed at identifying changes in the clones causing iGAS in Portugal. Antimicrobial susceptibility testing, emm typing and superantigen (SAg) gene profiling were performed for 381 iGAS isolates from 2010–2015. Macrolide resistance decreased to 4%, accompanied by the disappearance of the M phenotype and an increase of the iMLSB phenotype. The dominant emm types were: emm1 (28%), emm89 (11%), emm3 (9%), emm12 (8%), and emm6 (7%). There were no significant changes in the prevalence of individual emm types, emm clusters, or SAg profiles when comparing to 2006–2009, although an overall increasing trend was recorded during 2000–2015 for emm1, emm75, and emm87. Short-term increases in the prevalence of emm3, emm6, and emm75 may have been driven by concomitant SAg profile changes observed within these emm types, or reflect the emergence of novel genomic variants of the same emm types carrying different SAgs.

Download Full-text

ORNITHINE DECARBOXYLASE GENE EXPRESSION IN HUMAN LUNG ADENOCARCINOMA CELL (A549) TREATED WITH POMEGRANATE JUICE

Jurnal Teknologi ◽

10.11113/jt.v77.6746 ◽

2015 ◽

Vol 77 (25) ◽

Author(s):

Radiah Abdul Ghani ◽

Nik Nurasyikin Nik Abdul Malek ◽

Najwa Farhah Md. Yusof ◽

Elyna Fatini Jamil

Keyword(s):

Gene Expression ◽

Lung Cancer ◽

Cell Growth ◽

Ornithine Decarboxylase ◽

Quantitative Polymerase Chain Reaction ◽

A549 Cells ◽

Cellular Growth ◽

Pomegranate Juice ◽

Trypan Blue Exclusion ◽

Cancer Management

The polyamine biosynthesis pathway plays a significant role in cell growth, both normal and malignant. As polyamines are crucial in cellular growth and differentiation, they are linked to the development of cancer, with higher polyamine level observed in cancerous cells than in healthy cells. Accordingly, suppressing the polyamine pathway has been found to disrupt tumour development. Chemoprevention is considered a more feasible option in cancer management than chemotherapy, with a focus on natural chemopreventive agent. Pomegranate is known to inhibit several progression of lung cancer, although prior studies on the chemopreventive effect of pomegranate on lung cancer did not explore into polyamine pathway. Hence, this study investigated the effect of pomegranate juice on the polyamine pathway, by focusing on the biosynthesis involving ornithine decarboxylase (ODC), the rate limiting enzyme in the pathway. Quantitative polymerase chain reaction (qPCR) was applied to quantify the changes in ODC gene expression in A549 cells treated with pomegranate. The inhibition of growth was determined using Trypan Blue exclusion and the changes in intracellular polyamine in pomegranate treated cells was observed using High Performance Liquid Chromatography (HPLC). It was found that there was inhibition of A549 cell growth and reduced in intracellular polyamine content in pomegranate treated cells. The ODC expression was significantly inhibited compared to untreated cells, with a 48-fold difference. While this finding supports the hypothesis, there is much yet to be elucidated regarding its exact mechanism.

Download Full-text