scholarly journals Nasty Prophages and the Dynamics of Antibiotic-Tolerant Persister Cells

2017 ◽  
Author(s):  
Alexander Harms ◽  
Cinzia Fino ◽  
Michael A. Sørensen ◽  
Szabolcs Semsey ◽  
Kenn Gerdes
Keyword(s):  
Antibiotic Treatment ◽  
Bacterial Growth ◽  
Culture Conditions ◽  
Growth Stages ◽  
Persister Cells ◽  
E Coli ◽  
Common Basis ◽  
Mutant Strains ◽  
Future Work

AbstractBacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drivesEscherichia colipersister formation through protease Lon and the activation of toxin-antitoxin (TA) modules. This model found support in the field, but also generated controversy as part of recent heated debates on the validity of significant parts of the literature. In this study, we therefore used our previous work as a model to critically examine common experimental procedures in order to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variation of culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage φ80, a notorious laboratory contaminant, has apparently caused several phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon, but do not anymore support a role of TA modules inE. colipersister formation. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.ImportanceThe recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies have identified multiple genetic pathways involved in persister formation, but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely different results based on ill-controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis of future studies on the formation and eradication of antibiotic-tolerant persisters.

scholarly journals Prophages and Growth Dynamics Confound Experimental Results with Antibiotic-Tolerant Persister Cells

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Alexander Harms ◽  
Cinzia Fino ◽  
Michael A. Sørensen ◽  
Szabolcs Semsey ◽  
Kenn Gerdes
Keyword(s):  
Antibiotic Treatment ◽  
Bacterial Growth ◽  
Growth Dynamics ◽  
Culture Conditions ◽  
Growth Stages ◽  
Persister Cells ◽  
E Coli ◽  
Common Basis ◽  
Mutant Strains ◽  
Future Work

ABSTRACTBacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drivesEscherichia colipersister formation through protease Lon and activation of toxin-antitoxin (TA) modules. This model found considerable support among researchers studying persisters but also generated controversy as part of recent debates in the field. In this study, we therefore used our previous work as a model to critically examine common experimental procedures to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variations in culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage ϕ80, a notorious laboratory contaminant, apparently caused several of the phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon but no longer support a role of TA modules inE. colipersister formation under unstressed conditions. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.IMPORTANCEThe recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies identified multiple genetic pathways involved in persister formation but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study, we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely differing results based on poorly controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis for future studies on the formation and eradication of antibiotic-tolerant persisters.

Transcriptional regulation of a hybrid cluster (prismane) protein

2005 ◽  
Vol 33 (1) ◽  
pp. 195-197 ◽  
Author(s):  
N.A. Filenko ◽  
D.F. Browning ◽  
J.A. Cole
Keyword(s):  
Physiological Function ◽  
Response Regulator ◽  
Anaerobic Growth ◽  
Hybrid Cluster ◽  
E Coli ◽  
Mutant Strains ◽  
Translation Initiation Codon ◽  
Nitric Oxide Reduction ◽  
Nitrate And Nitrite

HCP (hybrid-cluster protein) contains two Fe/S clusters, one of which is a hybrid [4Fe-2S-2O] cluster. Despite intensive study, its physiological function has not been reported. The Escherichia coli hcp gene is located in a two-gene operon with hcr, which encodes an NADH-dependent HCP reductase. E. coli HCP is detected after anaerobic growth with nitrate or nitrite: possible roles for it in hydroxylamine or nitric oxide reduction have been proposed. To study the regulation and role of HCP, an hcp::lacZ fusion was constructed and transformed into fnr, arcA and norR mutant strains of E. coli. Transcription from the hcp promoter was induced during anaerobic growth. Only the fnr mutant was defective in hcp expression. Nitrate- and nitrite-induced transcription from the hcp promoter was activated by the response regulator proteins NarL and NarP. Gel retardation assays were used to show that FNR (fumarate-nitrate regulation) and NarL form a complex with the hcp promoter. Transcription of the hcp-hcr operon initiates at a thymine nucleotide located 31 bp upstream of the translation-initiation codon. HCP has been overexpressed from a recombinant plasmid for physiological studies.

Molecular Cloning of the gyrA andgyrB Genes of Bacteroides fragilis Encoding DNA Gyrase

1999 ◽  
Vol 43 (10) ◽  
pp. 2423-2429 ◽  
Author(s):  
Yoshikuni Onodera ◽  
Kenichi Sato
Keyword(s):  
Hot Spot ◽  
Bacteroides Fragilis ◽  
Dna Gyrase ◽  
E Coli ◽  
Important Target ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Gyrase A ◽  
Selection For

ABSTRACT The genes encoding the DNA gyrase A and B subunits ofBacteroides fragilis were cloned and sequenced. ThegyrA and gyrB genes code for proteins of 845 and 653 amino acids, respectively. These proteins were expressed inEscherichia coli, and the combination of GyrA and GyrB exhibited ATP-dependent supercoiling activity. To analyze the role of DNA gyrase in quinolone resistance of B. fragilis, we isolated mutant strains by stepwise selection for resistance to increasing concentrations of levofloxacin. We analyzed the resistant mutants and showed that Ser-82 of GyrA, equivalent to resistance hot spot Ser-83 of GyrA in E. coli, was in each case replaced with Phe. These results suggest that DNA gyrase is an important target for quinolones in B. fragilis.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

THE ROLE OF PROTON ATPase SPECIFIC INHIBITOR $N,N'$-DICYCLOHEXYLCARBODIIMIDE AND EXTERNAL FORMATE CONCENTRATION ON $E.~COLI$ GROWTH DURING MIXED CARBON SOURCES FERMENTATION AT DIFFERENT pHs.

2021 ◽  
Vol 55 (1 (254)) ◽  
pp. 67-74
Author(s):  
Heghine Kh. Gevorgyan ◽  
Anait V. Vassilian ◽  
Karen A. Trchounian
Keyword(s):  
Regulatory Protein ◽  
Carbon Sources ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
E Coli ◽  
Proton Atpase ◽  
Mutant Strains ◽  
Atpase Inhibition ◽  
Compensatory Effect

This research is focused on the investigation of specific growth rate changes of $E.~coli$ wild type and mutant strains with defect of Hyd, FDH enzymes and FhlA regulatory protein in the presence of $N,N'$-dicyclohexylcarbodiimide (DCCD) and external formate various concentration during co-fermentation of glucose, glycerol and formate at pHs $5.5-7.5.$ The highest value of SGR was observed at pH 7.5. It was revealed that SGR depends on external formate concentration at all pHs. DCCD inhibitory effect was shown mainly at pH 7.5 and partially at pH 6.5 and 5.5. In the case of the F0F1-ATPase inhibition FhlA compensatory effect on SGR was revealed.

scholarly journals Macrophage Class A Scavenger Receptor-Mediated Phagocytosis of Escherichia coli: Role of Cell Heterogeneity, Microbial Strain, and Culture Conditions In Vitro

2000 ◽  
Vol 68 (4) ◽  
pp. 1953-1963 ◽  
Author(s):  
Leanne Peiser ◽  
Peter J. Gough ◽  
Tatsuhiko Kodama ◽  
Siamon Gordon
Keyword(s):  
Escherichia Coli ◽  
Host Defense ◽  
Bacterial Strain ◽  
Chinese Hamster Ovary Cells ◽  
Culture Conditions ◽  
E Coli ◽  
Class A

ABSTRACT Macrophage class A scavenger receptors (SR-AI and SR-AII) contribute to host defense by binding polyanionic ligands such as lipopolysaccharide and lipoteichoic acid. SR-A knockout (SR-A−/−) mice are more susceptible to endotoxic shock and Listeria monocytogenes infection in vivo, possibly due to decreased clearance of lipopolysaccharide and microorganisms, respectively. We have used flow cytometry to analyze the role of SR-A and other scavenger-like receptors in phagocytosis of bacteria in vitro. Chinese hamster ovary cells stably transfected with human SR-A bound Escherichia coli and Staphylococcus aureus but ingested few organisms. Primary human monocyte-derived macrophages (Mφ) bound and ingested E. coli more efficiently, and this was partially but selectively blocked by the general SR inhibitor, poly(I). A specific and selective role for SR-A was shown, since bone marrow culture-derived Mφ from SR-A−/− mice ingested fewer E. coli organisms than did wild-type cells, while uptake of antibody-opsonized E. coli was unaffected. SR-A-dependent uptake of E. colivaried with the bacterial strain; ingestion of DH5α and K1 by SR-A−/− Mφ was reduced by 30 to 60% and 70 to 75%, respectively. Phagocytosis and endocytosis via SR-A were markedly down-modulated when Mφ were plated on serum-coated tissue culture plastic compared to bacteriologic plastic, where cell adhesion is mediated by SR-A and CR3, respectively. This paper demonstrates that SR-A can bind and ingest bacteria directly, consistent with a role in host defense in vivo, and highlights the importance of the source of the Mφ, bacterial strain, and culture conditions on receptor function in vitro.

scholarly journals Crucial Role of ppGpp in the Resilience of Escherichia coli to Growth Disruption

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
pp. e01132-20
Author(s):  
Clément Patacq ◽  
Nicolas Chaudet ◽  
Fabien Létisse
Keyword(s):  
Escherichia Coli ◽  
Bacterial Growth ◽  
Crucial Role ◽  
Stringent Response ◽  
Content Type ◽  
E Coli ◽  
Changing Environments ◽  
Growth Recovery ◽  
Growth Disruption

ABSTRACTBacteria grow in constantly changing environments that can suddenly become completely depleted of essential nutrients. The stringent response, a rewiring of the cellular metabolism mediated by the alarmone (p)ppGpp, plays a crucial role in adjusting bacterial growth to the severity of the nutritional stress. The ability of (p)ppGpp to trigger a slowdown of cell growth or induce bacterial dormancy has been widely investigated. However, little is known about the role of (p)ppGpp in promoting growth recovery after severe growth inhibition. In this study, we performed a time-resolved analysis of (p)ppGpp metabolism in Escherichia coli as it recovered from a sudden slowdown in growth. The results show that E. coli recovers by itself from the growth disruption provoked by the addition of serine hydroxamate, the serine analogue that we used to induce the stringent response. Growth inhibition was accompanied by a severe disturbance of metabolic activity and, more surprisingly, a transient overflow of valine and alanine. Our data also show that ppGpp is crucial for growth recovery since in the absence of ppGpp, E. coli’s growth recovery was slower. In contrast, an increased concentration of pppGpp was found to have no significant effect on growth recovery. Interestingly, the observed decrease in intracellular ppGpp levels in the recovery phase correlated with bacterial growth, and the main effect involved in the return to the basal level was identified by flux calculation as growth dilution. This report thus significantly expands our knowledge of (p)ppGpp metabolism in E. coli physiology.IMPORTANCE The capacity of microbes to resist and overcome environmental insults, known as resilience, allows them to survive in changing environments but also to resist antibiotic and biocide treatments and immune system responses. Although the role of the stringent response in bacterial resilience to nutritional stresses has been well studied, little is known about its importance in the ability of the bacteria to not just resist but also recover from these disturbances. To address this important question, we investigated growth disruption resilience in the model bacterium Escherichia coli and its dependence on the stringent response alarmone (p)ppGpp by quantifying ppGpp and pppGpp levels as growth was disrupted and then recovered. Our findings may thus contribute to understanding how ppGpp improves E. coli’s resilience to nutritional stress and other environmental insults.

25 Role of the urinalysis in managing infants less than 60 days old with a positive urine culture

2021 ◽  
Vol 26 (Supplement_1) ◽  
pp. e18-e19
Author(s):  
Steffany Charles ◽  
Marissa Aycan ◽  
Michelle D’Alessandro ◽  
Andrew Latchman ◽  
Gita Wahi
Keyword(s):  
Blood Cell ◽  
Antibiotic Treatment ◽  
Urine Culture ◽  
Positive Urine Culture ◽  
Blood Cell Count ◽  
Intravenous Antibiotic ◽  
Biochemical Characteristics ◽  
E Coli ◽  
Positive Urine

Abstract Primary Subject area Hospital Paediatrics Background Urinary tract infections (UTIs) in infants less than 60 days of age, often lead to hospitalization and intravenous antibiotic treatment. There are currently no national recommendations for the diagnosis and management of UTIs in this population. Historically, the urine culture alone was thought to be sufficient for diagnosing UTI due to concerns regarding the sensitivity of urinalysis (UA) in young infants. However, there is evidence that in children of all ages, the absence of pyuria in children with UTIs is rare. The role of the UA in influencing the current management of infants aged less than 60 days, with a positive urine culture, remains unclear. Objectives The objective of this study was to describe the characteristics of infants aged less than 60 days old with a positive urine culture. Specifically, we sought to: 1) determine the differences in clinical and biochemical characteristics of infants with a positive UA versus a negative UA; and 2) determine if UA result was associated with length of intravenous antibiotic treatment. Design/Methods We completed a retrospective cohort study of infants aged 0 to 60 days with a positive urine culture, cared for in a Canadian tertiary care children’s hospital between April 2016 and March 2019. Patients with a history of prematurity and those with a concomitant bacteremia or meningitis were excluded. A positive urine culture was defined as greater than 10,000 CFU/ml of a single organism obtained from a catheter specimen. A positive UA was defined by positive nitrites and/or a positive leukocyte esterase screen, and/or pyuria (greater than 5 white blood cells per high power field). Clinical and biochemical characteristics of infant groups with a positive UA, versus negative UA, were compared. Linear regression analysis was used to understand the association between UA result and length of intravenous antibiotic treatment. Results A total of 718 charts were screened. Eighty-nine patients met inclusion criteria, of which 52 (58%) had a positive UA and 37 (42%) had a negative UA. Of the 89 included infants, the average age was 32.5 days (SD 16.7), 31.5% were female, 79.8% had fever at presentation, and the average length of stay was 5.6 days (SD 2.9). Patients with a positive UA were more often febrile at presentation (90.3% vs. 64.9%, p = 0.006), and had a higher CRP (51.0, SD 56.1 vs. 8.1, SD 13.0), p < 0.001), higher white blood cell count (15.2, SD 6.0 vs. 11.0, SD 5.7, p= 0.002), and more often had urine culture growth of E. Coli (96.2% vs. 62.9%, p < 0.001). UA result was not associated with length of intravenous antibiotic treatment (p= 0.15). Conclusion This study demonstrates that infants with a positive urine culture and a positive UA result had more characteristics of infection, including fever, elevated CRP, elevated white blood cell count, and growth of E. Coli uropathogen, compared to infants with a negative UA. Despite this difference, there was no significant difference in length of intravenous antibiotic treatment between UA-positive versus UA-negative groups. This study suggests that infants with clinical and biochemical findings supportive of a UTI more often had a positive UA, along with a positive culture, and that the UA should be used as a screening test for UTI in infants aged less than 60 days.

Impact of bile duct obstruction on hepatic E. coli infection: role of IL-10

2006 ◽  
Vol 291 (1) ◽  
pp. G91-G94 ◽  
Author(s):  
D. Rohan Jeyarajah ◽  
Mariusz L. Kielar ◽  
Hoosein Saboorian ◽  
Prameela Karimi ◽  
Nicole Frantz ◽  
...  
Keyword(s):  
Bile Duct ◽  
Bacterial Growth ◽  
Biliary Obstruction ◽  
Bile Duct Ligation ◽  
Hepatic Clearance ◽  
Hepatic Necrosis ◽  
E Coli ◽  
Protein Levels ◽  
Duct Ligation

Biliary obstruction in the setting of hepatic bacterial infection has great morbidity and mortality. We developed a novel murine model to examine the effect of biliary obstruction on the clearance of hepatic Escherichia coli infection. This model may allow us to test the hypothesis that biliary obstruction itself adversely affects clearance of hepatic infections even if the bacteria are introduced into the liver by a nonbiliary route. We ligated the bile ducts of C57BL/6 mice on days − 1, 0, or + 1, relative to a day 0 portal venous injection of E. coli. We monitored survival, hepatic bacterial growth, pathology, and IL-10 protein levels. The role of IL-10 in this model was further examined using IL-10 knockout mice. Mice with bile duct ligation at day +1 or 0, relative to portal venous infection at day 0, had decreased survival compared with mice with only portal venous infection. The impaired survival was associated with greater hepatic bacterial growth, hepatic necrosis, and increased production of IL-10. Interestingly, the transgenic knockout of IL-10 resulted in impaired survival in mice with bile duct ligation and portal venous infection. Biliary obstruction had a dramatic detrimental effect on hepatic clearance of portal venous E. coli infection. This impaired clearance is associated with increased IL-10 production. However, transgenic knockout of IL-10 increased mortality after hepatic infection.

scholarly journals Crucial role of ppGpp in the resilience of Escherichia coli to growth disruption

2020 ◽  
Author(s):  
Clément Patacq ◽  
Nicolas Chaudet ◽  
Fabien Letisse
Keyword(s):  
Escherichia Coli ◽  
Growth Inhibition ◽  
Bacterial Growth ◽  
Crucial Role ◽  
Stringent Response ◽  
E Coli ◽  
Changing Environments ◽  
Growth Recovery ◽  
Growth Disruption

ABSTRACTBacteria grow in constantly changing environments that can suddenly become completely deleted in essential nutrients. The stringent response, a rewiring of the cellular metabolism mediated by the alarmone (p)ppGpp, plays a crucial role in adjusting bacterial growth to the severity of the nutritional insult. The ability of (p)ppGpp to trigger a slowdown of cell growth or induce bacterial dormancy has been widely investigated. However, little is known about the role of (p)ppGpp in promoting growth recovery after severe growth inhibition. In this study, we performed a time-resolved analysis of (p)ppGpp metabolism in Escherichia coli as it recovered from a sudden slowdown in growth. Results show that E. coli recovers by itself from the growth disruption provoked by the addition of serine hydroxamate, the serine analogue that we used to induce the stringent response. Growth inhibition was accompanied by a severe disturbance of metabolic activity and more surprisingly, by a transient overflow of valine and alanine. Our data also show that ppGpp is crucial for growth recovery since in the absence of ppGpp, E. coli’s growth recovery was slower. In contrast, an increased concentration of pppGpp was found to have no significant effect on growth recovery. Interestingly, the observed decrease in intracellular ppGpp levels in the recovery phase correlated with bacterial growth and the main effect involved was identified as growth dilution rather than active degradative process. This report thus significantly expands our knowledge of (p)ppGpp metabolism in E. coli physiology.IMPORTANCEThe capacity of microbes to resist and overcome environmental insults, know as resilience, allows them to survive in changing environments but also to resist antibiotic and biocide treatments, immune system responses. Although the role of the stringent response in bacterial resilience to nutritional insults has been well studied, little is known about its importance in the ability of the bacteria to not just resist but also recover from these disturbances. To address this important question, we investigated growth disruption resilience in the model bacterium Escherichia coli and its dependency on the stringent response alarmone (p)ppGpp by quantifying ppGpp and pppGpp levels as growth was disrupted and then recovered. Our findings may thus contribute to understanding how ppGpp improves E. coli’s resilience to nutritional stress and other environmental insults.

Sign in / Sign up

Export Citation Format

Share Document