Non-growing Bacteria Research Articles

Potential for repurposing the personal care product preservatives bronopol and bronidox as broad-spectrum antibiofilm agents for topical application.

Bacterial biofilms represent a major impediment to healing in chronic wounds and are largely refractory to the antibacterial agents currently used in wound management. From a repurposing screen of compounds considered safe for topical application in humans, we report the identification of the personal care product preservatives bronopol and bronidox as broad-spectrum antibiofilm agents and potential candidates for reducing biofilm burden in chronic wounds. Antibiofilm activity was assessed by viable counting against single-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa in the Calgary Biofilm Device, and against mixed-species biofilms of the two organisms growing on nitrocellulose discs. Bronopol and bronidox exhibited broad-spectrum antibiofilm activity that encompassed the two major wound pathogens, S. aureus and P. aeruginosa. When impregnated into gauze dressings at their existing maximum authorized concentrations for safe use and placed onto an established mixed-species biofilm, bronopol and bronidox completely eradicated P. aeruginosa and achieved an ∼5 log10 reduction in the S. aureus population. The antibiofilm action of bronopol and bronidox was attributed to their ability to kill slow- or non-growing bacteria found in biofilms, and both compounds exhibited synergistic antibiofilm effects in combination with established wound-treatment agents. Bronopol and bronidox kill bacteria regardless of growth state, a property that endows them with broad-spectrum antibiofilm activity. As this effect is observed at concentrations authorized for use on human skin, these compounds represent promising candidates for the treatment of chronic wounds.

In vitro activity of neem (Azadirachta indica) oil extract against Helicobacter pylori

Ethnopharmacological relevanceThe neem tree (Azadirachta indica A.Juss), of the Meliaceae family, has been used in India for millennia in traditional medicine. Parts of the tree are used to treat problems with the gastrointestinal tract, urinary tract, and hair; to combat infections of smallpox and plasmodium; and to treat ulcers, diabetes, blood pressure, headache, and heartburn. Natural products and extracts from the tree have been reported to have antimicrobial, antifungal, and antiparasitic activities. Aim of the studyAntibiotic resistance in the gastric pathogen Helicobacter pylori is increasing, and novel therapeutics to eradicate this bacterium are needed. Given the growing interest in the use of natural products as antimicrobials, this study was designed to examine the bactericidal effects of an extract of neem oil against H. pylori. Materials and methodsNeem oil was obtained from a commercial source and subjected to liquid-liquid extraction with diethyl ether and aqueous methanol; the methanol-soluble fraction was retained. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined against nine strains of H. pylori. Additionally, specific properties of the extract were characterized using H. pylori strain G27: bactericidal kinetics, reversibility, and effectiveness under growth arrest conditions and at low pH. The hemolytic activity of the extract was measured in vitro. ResultsThe MIC and MBC of the extract against the H. pylori strains were between 25 and 51 µg/mL and 43–68 µg/mL, respectively. The bactericidal activity was time- and concentration-dependent, and at the highest concentrations (75–105 µg/mL), no detectable bacteria were present by 6 h. The activity of the extract was reversible, independent of H. pylori growth, and increased at low pH. The extract exhibited no appreciable hemolytic activity. ConclusionsNeem oil extract has significant bactericidal activity against H. pylori. The extract has several favorable pharmacological properties, including ability to kill non-growing bacteria, increased activity at low pH, and no hemolytic activity. The compound(s) present in the extract could potentially be used as a future treatment for H. pylori infection.

Physicochemical Factors That Favor Conjugation of an Antibiotic Resistant Plasmid in Non-growing Bacterial Cultures in the Absence and Presence of Antibiotics.

Horizontal gene transfer (HGT) of antibiotic resistance genes has received increased scrutiny from the scientific community in recent years owing to the public health threat associated with antibiotic resistant bacteria. Most studies have examined HGT in growing cultures. We examined conjugation in growing and non-growing cultures of E. coli using a conjugative multi antibiotic and metal resistant plasmid to determine physiochemical parameters that favor horizontal gene transfer. The conjugation frequency in growing and non-growing cultures was generally greater under shaken than non-shaken conditions, presumably due to increased frequency of cell collisions. Non-growing cultures in 9.1 mM NaCl had a similar conjugation frequency to that of growing cultures in Luria-Bertaini broth, whereas those in 1 mM or 90.1 mM NaCl were much lower. This salinity effect on conjugation was attributed to differences in cell-cell interactions and conformational changes in cell surface macromolecules. In the presence of antibiotics, the conjugation frequencies of growing cultures did not increase, but in non-growing cultures of 9.1 mM NaCl supplemented with Cefotaxime the conjugation frequency was as much as nine times greater than that of growing cultures. The mechanism responsible for the increased conjugation in non-growing bacteria was attributed to the likely lack of penicillin-binding protein 3 (the target of Cefotaxime), in non-growing cells that enabled Cefotaxime to interact with the plasmid and induce conjugation. Our results suggests that more attention may be owed to HGT in non-growing bacteria as most bacteria in the environment are likely not growing and the proposed mechanism for increased conjugation may not be unique to the bacteria/plasmid system we studied.

Discovery and Optimization of nTZDpa as an Antibiotic Effective Against Bacterial Persisters.

Conventional antibiotics are not effective in treating infections caused by drug-resistant or persistent nongrowing bacteria, creating a dire need for the development of new antibiotics. We report that the small molecule nTZDpa, previously characterized as a nonthiazolidinedione peroxisome proliferator-activated receptor gamma partial agonist, kills both growing and persistent Staphylococcus aureus cells by lipid bilayer disruption. S.aureus exhibited no detectable development of resistance to nTZDpa, and the compound acted synergistically with aminoglycosides. We improved both the potency and selectivity of nTZDpa against MRSA membranes compared to mammalian membranes by leveraging synthetic chemistry guided by molecular dynamics simulations. These studies provide key insights into the design of selective and potent membrane-active antibiotics effective against bacterial persisters.

Phenotypic signatures and genetic determinants of oxacillin tolerance in a laboratory mutant of Staphylococcus aureus.

Addition of β-lactam antibiotics to growing cultures of bacteria inhibit synthesis of the bacterial cell wall peptidoglycan accompanied by killing (loss of viable titer) and lysis (physical disintegration) of the cells. However, it has also been well established that these antibiotics are not effective in killing non-growing or slow-growing bacteria and the mechanism of this “antibiotic tolerance” is not well understood. In this study, we report on the genetic basis and phenotypic properties of an antibiotic tolerant derivative of the methicillin susceptible S. aureus strain 27s. Cultures were exposed to “pulses” of high concentrations of oxacillin followed by outgrowth of the surviving bacteria. This procedure quickly selected for antibiotic tolerant mutants with an increased ability to survive antibiotic treatment without increase in the MIC value for the antibiotic. Such mutants also exhibited longer lag phase, decreased lysis, virtually no change in antibiotic susceptibilities, cross tolerance to D-cycloserine and vancomycin, and increase in biofilm formation in the presence of high concentrations of oxacillin. Whole genome sequencing showed that these altered properties were linked to mutations in the atl and gdpP genes.

Activity of acetyltransferase toxins involved in Salmonella\xa0persister formation during macrophage infection

Non-typhoidal Salmonella strains are responsible for invasive infections associated with high mortality and recurrence in sub-Saharan Africa, and there is strong evidence for clonal relapse following antibiotic treatment. Persisters are non-growing bacteria that are thought to be responsible for the recalcitrance of many infections to antibiotics. Toxin–antitoxin systems are stress-responsive elements that are important for Salmonella persister formation, specifically during infection. Here, we report the analysis of persister formation of clinical invasive strains of Salmonella Typhimurium and Enteritidis in human primary macrophages. We show that all the invasive clinical isolates of both serovars that we tested produce high levels of persisters following internalization by human macrophages. Our genome comparison reveals that S. Enteritidis and S. Typhimurium strains contain three acetyltransferase toxins that we characterize structurally and functionally. We show that all induce the persister state by inhibiting translation through acetylation of aminoacyl-tRNAs. However, they differ in their potency and target partially different subsets of aminoacyl-tRNAs, potentially accounting for their non-redundant effect.

Molecular Basis of Bacterial Longevity.

ABSTRACTIt is well known that many bacteria can survive in a growth-arrested state for long periods of time, on the order of months or even years, without forming dormant structures like spores or cysts. How is such longevity possible? What is the molecular basis of such longevity? Here we used the Gram-negative phototrophic alphaproteobacterium Rhodopseudomonas palustris to identify molecular determinants of bacterial longevity. R. palustris maintained viability for over a month after growth arrest due to nutrient depletion when it was provided with light as a source of energy. In transposon sequencing (Tn-seq) experiments, we identified 117 genes that were required for long-term viability of nongrowing R. palustris cells. Genes in this longevity gene set are annotated to play roles in a number of cellular processes, including DNA repair, tRNA modification, and the fidelity of protein synthesis. These genes are critically important only when cells are not growing. Three genes annotated to affect translation or posttranslational modifications were validated as bona fide longevity genes by mutagenesis and complementation experiments. These genes and others in the longevity gene set are broadly conserved in bacteria. This raises the possibility that it will be possible to define a core set of longevity genes common to many bacterial species.

Lethal Effect of Photodynamic Treatment on Persister Bacteria.

Persister bacteria tolerate bactericidal antibiotics due to transient and reversible phenotypic changes. As these bacteria can limit the effectiveness of antibiotics to eradicate certain infections, their elimination is a relevant issue. Photodynamic therapy seems suitable for this purpose, but phenotypic tolerance to it has also been reported for Pseudomonas aeruginosa. To test whether any phenotypic feature could confer tolerance against both antibiotics and photoinactivation, survivors from exposures to light in the presence of methylene blue were treated with ofloxacin, an antibiotic effective on nongrowing bacteria. Susceptibility to ofloxacin was normal in these bacteria in spite of their increased ability to survive photodynamic inactivation, suggesting the absence of cross-tolerance. It thus seemed possible to use one of these treatments to eliminate bacteria which had phenotypic tolerance to the other. To test this strategy, persister bacteria emerging from ofloxacin treatments were submitted to the action of light and methylene blue while the antibiotic remained in the bacterial suspension. Persisters lost their clonogenic ability under these conditions and the effects of the treatments seemed to be synergistic. These observations suggest that photodynamic antimicrobial therapy could be used as a complement to antibiotic treatments to eliminate persister bacteria from localized infections.

Stationary-phase Mutagenesis Soft-agar Overlay Assays in Bacillus subtilis.

Elucidating how a population of non-growing bacteria generates mutations improves our understanding of phenomena like antibiotic resistance, bacterial pathogenesis, genetic diversity and evolution. To evaluate mutations that occur in nutritionally stressed non-growing bacteria, we have employed the strain B. subtilis YB955, which measures the reversions rates to the chromosomal auxotrophies hisC952, metB5 and leuC427 (Sung and Yasbin, 2002). This gain-of-function system has successfully allowed establishing the role played by repair systems and transcriptional factors in stress-associated mutagenesis (SPM) (Barajas- Ornelas et al., 2014 ; Gómez- Marroquín et al., 2016 ). In a recent study (Castro- Cerritos et al., 2017 ), it was found that Ribonucleotide Reductase (RNR) was necessary for SPM; this enzyme is essential in this bacterium. We engineered a conditional mutant of strain B. subtilis YB955 in which expression of the nrdEF operon was modulated by isopropyl-β-D-thiogalactopyranoside (IPTG) (Castro- Cerritos et al., 2017 ). The conditions to determine mutation frequencies conferring amino acid prototrophy in three genes (hisC952, metB5, leuC427) under nutritional stress in this conditional mutant are detailed here. This technique could be used to evaluate the participation of essential genes in the mutagenic processes occurring in stressed B. subtilis cells.

Single-cell RNA-seq ties macrophage polarization to growth rate of intracellular Salmonella.

Intracellular bacterial pathogens can exhibit large heterogeneity in growth rate inside host cells, with major consequences for the infection outcome. If and how the host responds to this heterogeneity remains poorly understood. Here, we combined a fluorescent reporter of bacterial cell division with single-cell RNA-sequencing analysis to study the macrophage response to different intracellular states of the model pathogen Salmonella enterica serovar Typhimurium. The transcriptomes of individual infected macrophages revealed a spectrum of functional host response states to growing and non-growing bacteria. Intriguingly, macrophages harbouring non-growing Salmonella display hallmarks of the proinflammatory M1 polarization state and differ little from bystander cells, suggesting that non-growing bacteria evade recognition by intracellular immune receptors. By contrast, macrophages containing growing bacteria have turned into an anti-inflammatory, M2-like state, as if fast-growing intracellular Salmonella overcome host defence by reprogramming macrophage polarization. Additionally, our clustering approach reveals intermediate host functional states between these extremes. Altogether, our data suggest that gene expression variability in infected host cells shapes different cellular environments, some of which may favour a growth arrest of Salmonella facilitating immune evasion and the establishment of a long-term niche, while others allow Salmonella to escape intracellular antimicrobial activity and proliferate.

Impact of pulsed light on cellular activity of Salmonella enterica.

The objective of this study was a comprehensive characterization of physiological changes of Salmonella enterica induced by intense broad spectrum pulsed light (PL). After exposing the bacteria to this nonthermal decontamination technology on a gel surface, multiple viability parameters beyond culturability were assessed. By applying flow cytometry, a luciferin-luciferase bioluminescence assay and a microplate assay to measure the current redox activity, the impact of pulsed light on the membrane potential, membrane integrity, esterase activity, efflux pump activity, expression of the green fluorescent protein (GFP), respiration activity and ATP-content of Salm.enterica ATCC BAA-1045 was determined. These culture-independent methods for assessing the bacterial activity were compared to the ability to grow on tryptic soy agar. It is shown that this strain is rather sensitive to PL considering colony count reductions, while on the other hand unculturable bacteria still exhibit significant cellular energetic functions. However, this residual activity after PL exposure significantly decreases during sample storage in buffer for 24h. This study also shows that the GFP expression of PL-treated cells which have rendered unculturable is severely reduced. This study reveals that although not all cellular functions of Salm.enterica are immediately shut down after PL exposure, the synthesis of new GFP is strongly reduced and affected to a similar extent as the culturability. It is shown for the first time, that even there is significant bacterial activity measurable after PL exposure, it is likely that nongrowing pathogenic bacteria like Salm.enterica are unable to express proteins, which is of great importance regarding their pathogenicity.

A Bacterial Component to Alzheimer's-Type Dementia Seen via a Systems Biology Approach that Links Iron Dysregulation and Inflammagen Shedding to Disease.

The progression of Alzheimer’s disease (AD) is accompanied by a great many observable changes, both molecular and physiological. These include oxidative stress, neuroinflammation, and (more proximal to cognitive decline) the death of neuronal and other cells. A systems biology approach seeks to organize these observed variables into pathways that discriminate those that are highly involved (i.e., causative) from those that are more usefully recognized as bystander effects. We review the evidence that iron dysregulation is one of the central causative pathway elements here, as this can cause each of the above effects. In addition, we review the evidence that dormant, non-growing bacteria are a crucial feature of AD, that their growth in vivo is normally limited by a lack of free iron, and that it is this iron dysregulation that is an important factor in their resuscitation. Indeed, bacterial cells can be observed by ultrastructural microscopy in the blood of AD patients. A consequence of this is that the growing cells can shed highly inflammatory components such as lipopolysaccharides (LPS). These too are known to be able to induce (apoptotic and pyroptotic) neuronal cell death. There is also evidence that these systems interact with elements of vitamin D metabolism. This integrative systems approach has strong predictive power, indicating (as has indeed been shown) that both natural and pharmaceutical iron chelators might have useful protective roles in arresting cognitive decline, and that a further assessment of the role of microbes in AD development is more than highly warranted.

A pharmacokinetic-pharmacodynamic model characterizing the emergence of resistant Escherichia coli subpopulations during ertapenem exposure.

Resistant subpopulations with reduced expression of outer membrane porins have been observed in ESBL-producing Escherichia coli during exposure to ertapenem. The aim of this work was to develop a pharmacokinetic-pharmacodynamic (PKPD) model to characterize the emergence of resistant E. coli during exposure to ertapenem and to predict bacterial killing following different dosing regimens of ertapenem. Data from in vitro time-kill experiments were used to develop a mechanism-based PKPD model for three E. coli strains: a native strain, an ESBL-producing strain, and an ESBL-producing strain with reduced expression of porins OmpF and OmpC. Each strain was exposed to static ertapenem concentrations (1-512 × MIC) for 24 h using starting inocula of ∼10(6) and 10(8) cfu/mL. The developed PKPD model consisted of three bacterial states: susceptible growing, less susceptible non-growing, and non-susceptible non-growing bacteria. A pre-existing bacterial subpopulation was used to describe the emergence of resistance. The PKPD model adequately characterized the data of the three E. coli strains investigated. Results from predictions suggest that the conventional dosage (1 g intravenously once daily) might result in regrowth of resistant subpopulations when used to treat infection caused by ESBL-producing strains. Resistant subpopulations frequently emerged in E. coli when exposed to ertapenem, supporting that the time course of emergence of resistance should be taken into consideration when selecting dosing regimens.

Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester.

Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single cell cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity. Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

A mechanism-based pharmacokinetic/pharmacodynamic model allows prediction of antibiotic killing from MIC values for WT and mutants.

In silico pharmacokinetic/pharmacodynamic (PK/PD) models can be developed based on data from in vitro time-kill experiments and can provide valuable information to guide dosing of antibiotics. The aim was to develop a mechanism-based in silico model that can describe in vitro time-kill experiments of Escherichia coli MG1655 WT and six isogenic mutants exposed to ciprofloxacin and to identify relationships that may be used to simplify future characterizations in a similar setting. In this study, we developed a mechanism-based PK/PD model describing killing kinetics for E. coli following exposure to ciprofloxacin. WT and six well-characterized mutants, with one to four clinically relevant resistance mutations each, were exposed to a wide range of static ciprofloxacin concentrations. The developed model includes susceptible growing bacteria, less susceptible (pre-existing resistant) growing bacteria, non-susceptible non-growing bacteria and non-colony-forming non-growing bacteria. The non-colony-forming state was likely due to formation of filaments and was needed to describe data close to the MIC. A common model structure with different potency for bacterial killing (EC50) for each strain successfully characterized the time-kill curves for both WT and the six E. coli mutants. The model-derived mutant-specific EC50 estimates were highly correlated (r(2) = 0.99) with the experimentally determined MICs, implying that the in vitro time-kill profile of a mutant strain is reasonably well predictable by the MIC alone based on the model.

Use of non-growing Lactococcus lactis cell suspensions for production of volatile metabolites with direct relevance for flavour formation during dairy fermentations.

BackgroundLactococcus lactis is a lactic acid bacterium that has been used for centuries in the production of a variety of cheeses, as these bacteria rapidly acidify milk and greatly contribute to the flavour of the fermentation end-products. After a short growth phase during cheese ripening L. lactis enters an extended non-growing state whilst still strongly contributing to amino acid-derived flavour formation. Here, a research approach is presented that allows investigation of strain- and amino acid-specific flavour formation during the non-growing state.ResultsNon-growing cells of five selected L. lactis strains were demonstrated to degrade amino acids into flavour compounds that are relevant in food fermentations and differs greatly from production of flavour compounds using growing cells. As observed earlier in other research set-ups and with other microorganisms, addition of NADH, α-ketoglutarate and pyridoxal-5-phosphate was demonstrated to be essential for optimal flavour formation, suggesting that intracellular pools of these substrates are too low for the significant production of the flavour compounds. Production of flavours during the non-growing phase strongly depends on the individual amino acids that were supplied, on the presence of other amino acids (mixtures versus single compounds), and on the strain used. Moreover, we observed that the plasmid-free model strains L. lactis MG1363 and IL1403 produce relatively low amounts of flavour components under the various conditions tested.ConclusionsBy using this simplified and rapid approach to study flavour formation by non-growing lactic acid bacteria, lengthy ripening periods are no longer required to assess the capacity of strains to produce flavours in the long, non-growing state of dairy fermentation. In addition, this method also provides insight into the conversion of single amino acids versus the conversion of a mixture of amino acids as produced during protein degradation. The generated results are complementary to earlier generated datasets using growing cells, allowing assessment of the full flavour forming potential of strains used as starter cultures in industrial food fermentation processes.

Comprehensive physicochemical, pharmacokinetic and activity profiling of anti-TB agents.

The discovery and development of TB drugs has met limited success, with two new drugs approved over the last 40 years. Part of the difficulty resides in the lack of well-established in vitro or in vivo targets of potency and physicochemical and pharmacokinetic parameters. In an attempt to benchmark and compare such properties for anti-TB agents, we have experimentally determined and compiled these parameters for 36 anti-TB compounds, using standardized and centralized assays, thus ensuring direct comparability across drugs and drug classes. Potency parameters included growth inhibition, cidal activity against growing and non-growing bacteria and activity against intracellular mycobacteria. Pharmacokinetic parameters included basic physicochemical properties, solubility, permeability and metabolic stability. We then attempted to establish correlations between physicochemical, in vitro and in vivo pharmacokinetic and pharmacodynamic indices to tentatively inform future drug discovery efforts. Two-thirds of the compounds tested showed bactericidal and intramacrophage activity. Most compounds exhibited favourable solubility, permeability and metabolic stability in standard in vitro pharmacokinetic assays. An analysis of human pharmacokinetic parameters revealed associations between lipophilicity and volume of distribution, clearance, plasma protein binding and oral bioavailability. Not surprisingly, most compounds with favourable pharmacokinetic properties complied with Lipinski's rule of five. However, most attempts to detect in vitro-in vivo correlations were unsuccessful, emphasizing the challenges of anti-TB drug discovery. The objective of this work is to provide a reference dataset for the TB drug discovery community with a focus on comparative in vitro potency and pharmacokinetics.

A Novel Antisense RNA from the Salmonella Virulence Plasmid pSLT Expressed by Non-Growing Bacteria inside Eukaryotic Cells

Bacterial small RNAs (sRNAs) are regulatory molecules playing relevant roles in response to environmental changes, stressful conditions and pathogenesis. The intracellular bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is known to regulate expression of some sRNAs during colonization of fibroblasts. Here, we characterize a previously unknown sRNA encoded in the S. Typhimurium pSLT virulence plasmid that is specifically up-regulated by non-growing dormant bacteria persisting inside fibroblasts. This sRNA was inferred in microarray expression analyses, which unraveled enhanced transcriptional activity in the PSLT047- PSLT046 (mig5) intergenic region. The sRNA transcript was further identified as a 597-nucleotide molecule, which we named IesR-1, for ‘Intracellular-expressed-sRNA-1′. IesR-1 expression is low in bacteria growing in axenic cultures across a variety of experimental conditions but displays a marked increase (∼200–300 fold) following bacterial entry into fibroblasts. Remarkably, induction of IesR-1 expression is not prominent in bacteria proliferating within epithelial cells. IesR-1 deletion affects the control of bacterial growth in defined fibroblast cell lines and impairs virulence in a mouse infection model. Expression analyses performed in the PSLT047-iesR-1-PSLT046 (mig5) region support a cis-acting regulatory mechanism of IesR-1 as antisense RNA over the PSLT047 transcript involving interaction at their respective 3′ ends and modulation of PSLT047 protein levels. This model is sustained by the scarce production of PSLT047 protein observed in non-growing intracellular bacteria and the high amount of PSLT047 protein produced by bacteria carrying a truncated IesR-1 version with separated 5′ and 3′ regions. Taken together, these data reveal that S. Typhimurium sRNAs encoded in the pSLT virulence plasmid respond to a state of persistence inside the host cell. As exemplified by IesR-1, some of these sRNAs may contribute to diminish the relative levels of proteins, such as PSLT047, which are probably dispensable for the intracellular lifestyle.

Dormant Intracellular Salmonella enterica Serovar Typhimurium Discriminates among Salmonella Pathogenicity Island 2 Effectors To Persist inside Fibroblasts

Salmonella enterica uses effector proteins delivered by type III secretion systems (TTSS) to colonize eukaryotic cells. Recent in vivo studies have shown that intracellular bacteria activate the TTSS encoded by Salmonella pathogenicity island-2 (SPI-2) to restrain growth inside phagocytes. Growth attenuation is also observed in vivo in bacteria colonizing nonphagocytic stromal cells of the intestinal lamina propria and in cultured fibroblasts. SPI-2 is required for survival of nongrowing bacteria persisting inside fibroblasts, but its induction mode and the effectors involved remain unknown. Here, we show that nongrowing dormant intracellular bacteria use the two-component system OmpR-EnvZ to induce SPI-2 expression and the PhoP-PhoQ system to regulate the time at which induction takes place, 2 h postentry. Dormant bacteria were shown to discriminate the usage of SPI-2 effectors. Among the effectors tested, SseF, SseG, and SseJ were required for survival, while others, such as SifA and SifB, were not. SifA and SifB dispensability correlated with the inability of intracellular bacteria to secrete these effectors even when overexpressed. Conversely, SseJ overproduction resulted in augmented secretion and exacerbated bacterial growth. Dormant bacteria produced other effectors, such as PipB and PipB2, that, unlike what was reported for epithelial cells, did not to traffic outside the phagosomal compartment. Therefore, permissiveness for secreting only a subset of SPI-2 effectors may be instrumental for dormancy. We propose that the S. enterica serovar Typhimurium nonproliferative intracellular lifestyle is sustained by selection of SPI-2 effectors that are produced in tightly defined amounts and delivered to phagosome-confined locations.

The mixotrophic protist Ochromonas danica is an indiscriminant predator whose fitness is influenced by prey type

Microbial predator-prey interactions are one of the primary trophic interactions linking biogeochemical cycles to ecosystem dynamics. The mixotrophic flagellate Ochromonas danica was used as a model predator to investigate feeding trends when supplied with actively growing and non-growing bacteria representing a variety of phylogenetic groups. The rate at which bacteria were ingested and the subsequent growth rate (ecological fitness) of O. danica were determined for each type of prey in each growth state. O. danica preferred bacteria between 0.6 and 1.0 μm 3 . It was, in general, an indiscriminate predator in that it readily ingested growing and non-growing bacteria spanning a range of phylogenetic groups, with some notable exceptions. Bacilli/Actinobacteria were ingested at significantly lower rates than Alpha- and Gamma-proteobacteria. There were no significant differences among rates at which Proteobacteria (Alpha-, Beta-, and Gamma-) were ingested. The ecological fitness of O. danica could not be predicted from ingestion rates. Predation on Bacilli/Actinobacteria resulted in growth rates significantly lower than when the flagellate preyed upon Gammaproteobacteria. Betaproteobacteria were readily ingested but always resulted in a significant decline in predator fitness. The data reveal that different types of bacteria have different nutritional value to a consumer. If other flagellates respond similarly, then it would seem that trophic transfer efficiency and nutrient regeneration depend on the diet-breadth of predators.