RpoS role in antibiotic resistance, tolerance and persistence in E. coli natural isolates.

BACKGROUND: The intrinsic concentration of RpoS, the second most abundant sigma factor, varies widely across the E. coli species. Bacterial isolates that express high levels of RpoS display high resistance to environmental stresses, such as temperature, pH and osmolarity shifts, but are less nutritional competent, making them less capable of utilising alternative nutrient sources. The role of RpoS in antibiotic resistance and persistence in standard laboratory domesticated strains has been examined in several studies, most demonstrating a positive role for RpoS. RESULTS: Using disk diffusion assays we examined bacterial resistance to 15 different antibiotics, including ß -lactams (penicillins, monobactams, carbapenems and cephalosporins), aminoglycosides, quinolones and anti-folates, in a representative collection of 328 E. coli natural isolates displaying a continuum of different levels of RpoS. There was an overall trend that isolates with higher levels of RpoS were slightly more resistant to these antibiotics. In addition, the effect of RpoS on bacterial tolerance and persistence to 3 different antibiotics - ampicillin, ciprofloxacin and kanamycin was evaluated through time-kill curves. Again, there was a small beneficial effect of RpoS on tolerance and persistence to these antibiotics, but this difference was not statistically significant. Finally, a K-12 strain expressing high levels of RpoS was compared with its isogenic RpoS-null counterpart, and no significant effect of RpoS was found. CONCLUSION: Based on a representative collection of the species E. coli, RpoS was found to have a very small impact on antibiotic resistance, tolerance, or persistence.

Bacterial toxin-antitoxin systems: Novel insights on toxin activation across populations and experimental shortcomings.

The alarming rise in hard-to-treat bacterial infections is of great concern to human health. Thus, the identification of molecular mechanisms that enable the survival and growth of pathogens is of utmost urgency for the development of more efficient antimicrobial therapies. In challenging environments, such as presence of antibiotics, or during host infection, metabolic adjustments are essential for microorganism survival and competitiveness. Toxin-antitoxin systems (TASs) consisting of a toxin with metabolic modulating activity and a cognate antitoxin that antagonizes that toxin are important elements in the arsenal of bacterial stress defense. However, the exact physiological function of TA systems is highly debatable and with the exception of stabilization of mobile genetic elements and phage inhibition, other proposed biological functions lack a broad consensus. This review aims at gaining new insights into the physiological effects of TASs in bacteria and exploring the experimental shortcomings that lead to discrepant results in TAS research. Distinct control mechanisms ensure that only subsets of cells within isogenic cultures transiently develop moderate levels of toxin activity. As a result, TASs cause phenotypic growth heterogeneity rather than cell stasis in the entire population. It is this feature that allows bacteria to thrive in diverse environments through the creation of subpopulations with different metabolic rates and stress tolerance programs.

Glyphosate affects persistence and tolerance but not antibiotic resistance.

Glyphosate is a herbicide widely used in food production that blocks the synthesis of aromatic amino acids in plants and in microorganisms and also induces the accumulation of the alarmone (p)ppGpp. The purpose of this study was to investigate whether glyphosate affects the resistance, tolerance or persistence of bacteria towards three different classes of antibiotics and the possible role of (p)ppGpp in this activity. Glyphosate did not affect the minimum inhibitory concentration of the tested antibiotics, but enhanced bacterial tolerance and/or persistence towards them. The upshift in ciprofloxacin and kanamycin tolerance was partially dependent on the presence of relA that promotes (p)ppGpp accumulation in response to glyphosate. Conversely, the strong increase in ampicillin tolerance caused by glyphosate was independent of relA. We conclude that by inducing aromatic amino acid starvation glyphosate contributes to the temporary increase in E. coli tolerance or persistence, but does not affect antibiotic resistance.

Cannabinoid-like meroterpenoids from Peperomia incana.

Ten previously undescribed metabolites were isolated from Peperomia incana (Haw.) A. Dietr. (Piperaceae), among which four contained a chromene moiety, two were identified as meroterpene lactones, and four were cannabinoid-like compounds. While the chemical structures of the compounds were assigned based on HRESIMS and 1D and 2D-NMR spectra analyses, the relative and absolute configurations were assigned from NOE correlations and a combination of ECD data and X-ray single crystal analyses, respectively. In a cytotoxic assay against a panel of seven human cancer cell lines (A549, MDA-MB-231, HeLa, DU 145, 5637, Hep G2, and MIA PaCa-2, which represent non-small cell lung cancer, as well as breast, cervical, prostate, bladder, liver, and pancreas carcinomas, respectively) most of the isolated compounds showed promising cytotoxic activities. The incanachromenes B, and incanabinoids A and C exhibited the highest cytotoxicity toward all tested cancer cell lines with IC50 values in the range of 5.0-10.0 µM, whereas incanolides A, B, and incanabinoid B showed the lowest cytotoxic activity. In addition, incanachromene C and incanabinoid C produced a significant antibacterial effect toward planktonic cells and biofilms of multidrug-resistant Staphylococcus aureus strains.

The Impact of the Highly Virulent SARS-CoV-2 Gamma Variant on Young Adults in the State of São Paulo: Was It Inevitable?

Background The coronavirus disease 2019 (COVID-19) pandemic had and is still having a tremendous impact on people all over the world, but it has been particularly harsh in South America. Nine out of 13 South American countries are among the 50 countries with the highest COVID-19 death rates. The gamma severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant that emerged by the end of 2020 in the Brazilian Amazon quickly spread throughout the country causing the harsh COVID-19 second wave. This variant displayed high viral loads, high transmissibility, and increased virulence as compared to previous variants. Aims The aim of this retrospective study is to revisit and analyse the epidemiology of the COVID-19 second wave in the state of São Paulo, the most populous Brazilian state. In addition to examining the possible factors that led to the emergence and propagation of the gamma variant, measures that could have prevented its spread and that of other highly virulent variants were also investigated. Materials and methods Data from São Paulo's official sources on morbidity, mortality, age distribution, and testing prior to and during the COVID-19 second wave (February - June 2021) and data regarding the distribution of SARS-CoV-2 variants in the country were parsed, analyzed, and compared to the period that anteceded the eruption of the second COVID-19 wave. Results In the state of São Paulo, the toll of the COVID-19 second wave surpassed that of the first 11 months of the pandemic (from March 2020 to January 2021), as 56% of the deaths occurred in the five months of the second wave between February and June 2021. The mean age of COVID-19 victims, which was already below life expectancy in the state dropped even further in the pandemic's second wave, reaching an average of 60 years of age. The years of life lost per death per month doubled and the case-fatality rate (CFR) of young adults (20-39 years old) more than trebled during this period. A number of hypotheses have been raised that might explain the emergence and spread of the gamma variant and the measures that could have been taken to prevent it and minimise its impact on the population. Conclusions Over 142,000 people died as a result of the SARS-CoV-2 gamma variant sweep in São Paulo in the first semester of 2021. Due to its high viral load, the gamma variant displayed high transmissibility and a high degree of virulence resulting in increased case fatality rates across most age tiers. Notably, this second wave was marked by a very significant increase in deaths among young adults. This increase was at least partially due to a deterioration in general health provoked by non-pharmaceutical interventions. In hindsight, a safer and more effective measure might have been to allow the free spread of the virus among the young and healthy in the first wave, thus conferring immunity against more virulent variants that emerged later on.

Correlation Between Mask Compliance and COVID-19 Outcomes in Europe.

Masking was the single most common non-pharmaceutical intervention in the course of the coronavirus disease 2019 (COVID-19) pandemic. Most countries have implemented recommendations or mandates regarding the use of masks in public spaces. The aim of this short study was to analyse the correlation between mask usage against morbidity and mortality rates in the 2020-2021 winter in Europe. Data from 35 European countries on morbidity, mortality, and mask usage during a six-month period were analysed and crossed. Mask usage was more homogeneous in Eastern Europe than in Western European countries. Spearman's correlation coefficients between mask usage and COVID-19 outcomes were either null or positive, depending on the subgroup of countries and type of outcome (cases or deaths). Positive correlations were stronger in Western than in Eastern European countries. These findings indicate that countries with high levels of mask compliance did not perform better than those with low mask usage.

A Broad Continuum of E. coli Traits in Nature Associated with the Trade-off Between Self-preservation and Nutritional Competence.

A trade-off between reproduction and survival is a characteristic of many organisms. In bacteria, growth is constrained when cellular resources are channelled towards environmental stress protection. At the core of this trade-off in Escherichia coli is RpoS, a sigma factor that diverts transcriptional resources towards general stress resistance. The constancy of RpoS levels in natural isolates is unknown. A uniform RpoS content in E. coli would impart a narrow range of resistance properties to the species, whereas a diverse set of RpoS levels in nature should result in a diverse range of stress susceptibilities. We explore the diversity of trade-off settings and phenotypes by measuring the level of RpoS protein in strains of E. coli cohabiting in a natural environment. Strains from a stream polluted with domestic waste were investigated in monthly samples. Analyses included E. coli phylogroup classification, RpoS protein level, RpoS-dependent stress phenotypes and the sequencing of rpoS mutations. The most striking finding was the continuum of RpoS levels, with a 100-fold range of RpoS amounts consistently found in individuals in the stream. Approximately 1.8% of the sampled strains carried null or non-synonymous mutations in rpoS. The natural isolates also exhibited a broad (>100-fold) range of stress resistance responses. Our results are consistent with the view that a multiplicity of survival-multiplication trade-off settings is a feature of the species E. coli. The phenotypic diversity resulting from the trade-off permits bet-hedging and the adaptation of E. coli strains to a very broad range of environments.

Bacteriophages as surrogates for the study of viral dispersion in open air.

The SARS-CoV-2 pandemic has revived the debate about the routes of virus transmission and their likelihoods. It is of utmost importance to assess the risks of contamination of susceptible people by infectious individuals and to evaluate the level of SARS-CoV-2 and other respiratory viruses transmission in the community. Most countries have imposed non-pharmaceutical measures to contain SARS-CoV-2 transmission, including physical distancing and mask wearing. Here we evaluated the spreading of viruses in open air using harmless Escherichia coli bacteriophages as a surrogate. Phages were sprayed towards Petri dishes seeded with bacteria at different lengths and angles. Our results showed that the transmission rate decreased exponentially with distance. The highest recorded transmission rate was [Formula: see text] PFU/plate when phages were sprayed from a 1 m distance, suggesting that the probability of transmission of a single virus at a 1 m distance is 1:100,000.

Competition for nutritional resources masks the true frequency of bacterial mutants.

BACKGROUND: It is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for selection. Unlike wild-type Escherichia coli, PHO-constitutive mutants overexpress alkaline phosphatase and hence can hydrolyze glycerol-2-phosphate (G2P) to glycerol and form colonies on plates having G2P as the sole carbon source. These mutations mostly occur in the pst operon. However, the frequency of PHO-constitutive colonies on the G2P selective plate is exceptionally low. RESULTS: We show that the rate in which spontaneous PHO-constitutive mutations emerge is about 8.0 × 10-6/generation, a relatively high rate, but the growth of most existing mutants is inhibited by their neighboring wild-type cells. This inhibition is elicited only by non-mutant viable bacteria that can take up and metabolize glycerol formed by the mutants. Evidence indicates that the few mutants that do form colonies derive from microclusters of mutants on the selective plate. A mathematical model that describes the fate of the wild-type and mutant populations under these circumstances supports these results. CONCLUSION: This scenario in which neither the wild-type nor the majority of the mutants are able to grow resembles an unavoidable "tragedy of the commons" case which results in the collapse of the majority of the population. Cooperation between rare adjacent mutants enables them to overcome the competition and eventually form mutant colonies. The inhibition of PHO-constitutive mutants provides an example of mutant frequency masked by orders of magnitude due to a competition between mutants and their ancestral wild-type cells. Similar "tragedy of the commons-like" cases may occur in other settings and should be taken into consideration while estimating true mutant frequencies and mutation rates.

In vivo Activity of Silver Nanoparticles Against Pseudomonas aeruginosa Infection in Galleria mellonella.

Pseudomonas aeruginosa is an opportunistic pathogen associated with life-threatening nosocomial and community-acquired infections. Antibiotic resistance is an immediate threat to public health and demands an urgent action to discovering new antimicrobial agents. One of the best alternatives for pre-clinical tests with animal models is the greater wax moth Galleria mellonella. Here, we evaluated the antipseudomonal activity of silver nanoparticles (AgNPs) against P. aeruginosa strain UCBPP-PA14 using G. mellonella larvae. The AgNPs were synthesized through a non-toxic biogenic process involving microorganism fermentation. The effect of AgNPs was assessed through characterization and quantification of the hemocytic response, nodulation and phenoloxidase cascade. On average, 80% of the larvae infected with P. aeruginosa and prophylactically treated with nanoparticles survived. Both the specific and total larvae hemocyte counts were restored in the treated group. In addition, the nodulation process and the phenoloxidase cascade were less exacerbated when the larvae were exposed to the silver nanoparticles. AgNPs protect the larvae from P. aeruginosa infection by directly killing the bacteria and indirectly by preventing an exacerbated immunological response against the pathogen. Our results suggest that the prophylactic use of AgNPs has a strong protective activity against P. aeruginosa infection.

Diversity in E. coli (p)ppGpp Levels and Its Consequences.

(p)ppGpp is at the core of global bacterial regulation as it controls growth, the most important aspect of life. It would therefore be expected that at least across a species the intrinsic (basal) levels of (p)ppGpp would be reasonably constant. On the other hand, the historical contingency driven by the selective pressures on bacterial populations vary widely resulting in broad genetic polymorphism. Given that (p)ppGpp controls the expression of many genes including those involved in the bacterial response to environmental challenges, it is not surprising that the intrinsic levels of (p)ppGpp would also vary considerably. In fact, null mutations or less severe genetic polymorphisms in genes associated with (p)ppGpp synthesis and hydrolysis are common. Such variation can be observed in laboratory strains, in natural isolates as well as in evolution experiments. High (p)ppGpp levels result in low growth rate and high tolerance to environmental stresses. Other aspects such as virulence and antimicrobial resistance are also influenced by the intrinsic levels of (p)ppGpp. A case in point is the production of Shiga toxin by certain E. coli strains which is inversely correlated to (p)ppGpp basal level. Conversely, (p)ppGpp concentration is positively correlated to increased tolerance to different antibiotics such as ß-lactams, vancomycin, and others. Here we review the variations in intrinsic (p)ppGpp levels and its consequences across the E. coli species.

Visible light plasmon excitation of silver nanoparticles against antibiotic-resistant Pseudomonas aeruginosa.

The interaction of metallic nanoparticles with light excites a local surface plasmon resonance (LSPR). This phenomenon enables the transfer of hot electrons to substrates that release Reactive Oxygen Species (ROS). In this context, the present study aimed at enhancing the antibacterial effect of citrate-covered silver nanoparticles (AgNPs) by LSPR excitation with visible LED. AgNPs possess excellent antimicrobial properties against Pseudomonas aeruginosa, one of the most refractory organisms to antibiotic treatment. The Minimum Inhibitory Concentration (MIC) of the AgNPs was 10 µg/ml under dark conditions and 5 µg/ml under light conditions. The combination of light and AgNPs led to 100% cell death after 60 min. Flow cytometry quantification showed that bacteria treated with LSPR-stimulated AgNPs displayed 4.8 times more ROS. This significant increase in ROS possibly accounts for most of the antimicrobial effect of the AgNPs. In addition, light exposition caused a small release of silver ions (0.4%) suggesting that silver ions may play a secondary role in P. aeruginosa death. Overall, the results presented here show that LSPR stimulation of AgNPs by visible light enhances the antimicrobial activity of silver nanoparticles and can be an alternative for the treatment of topic infections caused by antibiotic-resistant bacteria such as P. aeruginosa.

Phosphate uptake by the phosphonate transport system PhnCDE.

BACKGROUND: Phosphate is a fundamental nutrient for all creatures. It is thus not surprising that a single bacterium carries different transport systems for this molecule, each usually operating under different environmental conditions. The phosphonate transport system of E. coli K-12 is cryptic due to an 8 bp insertion in the phnE ORF. RESULTS: Here we report that an E. coli K-12 strain carrying the triple knockout ΔpitA Δpst Δugp reverted the phnE mutation when plated on complex medium containing phosphate as the main phosphorus source. It is also shown that PhnCDE takes up orthophosphate with transport kinetics compatible with that of the canonical transport system PitA and that Pi-uptake via PhnCDE is sufficient to enable bacterial growth. Ugp, a glycerol phosphate transporter, is unable to take up phosphate. CONCLUSIONS: The phosphonate transport system, which is normally cryptic in E. coli laboratory strains is activated upon selection in rich medium and takes up orthophosphate in the absence of the two canonical phosphate-uptake systems. Based on these findings, the PhnCDE system can be considered a genuine phosphate transport system.

Glyphosate induces the synthesis of ppGpp.

Glyphosate, the most widely used herbicide in both agricultural and urban areas is toxic for plants and for many bacterial species. The mechanism of action of glyphosate is through the inhibition of the EPSP synthase, a key enzyme in the biosynthetic pathway of aromatic amino acids. Here we show that glyphosate induces the stringent response in Escherichia coli. Bacteria treated with glyphosate stop growing and accumulate ppGpp. Both growth arrest and ppGpp accumulation are restored to normal levels upon addition of aromatic amino acids. Glyphosate-induced ppGpp accumulation is dependent on the presence of the (p)ppGpp synthetase RelA. However, unlike other cases of amino acid starvation, pppGpp could not be discerned. In a gppA background both ppGpp and pppGpp accumulated when exposed to glyphosate. Conversely, the wild-type strain and gppA mutant treated with serine hydroxamate accumulated high levels of both ppGpp and pppGpp. Altogether, the data indicate that glyphosate induces amino acid starvation resulting in a moderate accumulation of ppGpp and a reversible stringent response.

Two Draft Genome Sequences of Chromobacterium violaceum Isolates from the Rio Negro.

The draft genome sequences of two Chromobacterium violaceum strains isolated from the Rio Negro are reported here. These bacteria carry most genetic systems associated with the production of bioactive compounds, but unlike other C. violaceum strains, they lack a dedicated operon for arsenic resistance.

RpoS role in virulence and fitness in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that afflicts infants in developing countries. The most important virulence trait of EPEC is its ability to intimately adhere to cells in the small intestine, and to elicit diarrhea. The alternative sigma factor RpoS is involved in the virulence of several bacterial species. RpoS coordinates the general stress response and accumulates in cells under stress or in the stationary phase. RpoS levels differ across E. coli strains. High-RpoS strains are highly resistant to environmental stresses, but usually display low nutritional competence, while low-RpoS strains show the opposite phenotype. Here we investigated whether RpoS plays a role in the virulence and fitness of two different EPEC strains, E2348/69 and LRT9. A rpoS null mutation had a small positive effect on LRT9 adherence to epithelial cells, but the expression of the EPEC adhesins BfpA and intimin was not significantly affected by the mutation. E2348/69 adherence was not significantly affected by the rpoS mutation. The intrinsic level of RpoS was higher in LRT9 than in E2348/69 while the latter adhered more strongly and expressed higher levels of the adhesin BfpA than the former. Knockout of rpoS strongly impaired resistance to oxidative, osmotic and acid stress in both E2348/69 and LRT9. However, strain E2348/69 was significantly more sensitive to oxidative stress than LRT9. Finally, competition assays showed that the rpoS mutant of LRT9 displayed higher fitness under continuous culture than its isogenic wild-type strain, while E2348/69 outcompeted its rpoS mutant. In conclusion, RpoS plays mostly a positive role in EPEC biology and at least in the case of strain E2348/69 it is not constrained by the trade-off between vegetative growth and stress resistance.

Ciprofloxacin-Mediated Mutagenesis Is Suppressed by Subinhibitory Concentrations of Amikacin in Pseudomonas aeruginosa.

Resistance to antibiotics is a global health problem. Activation of the SOS response, and the subsequent elevation in mutagenesis, contributes to the appearance of resistance mutations. Among currently used drugs, quinolones are the most potent inducers of the SOS response. In the present study, we show that amikacin inhibits ciprofloxacin-mediated SOS induction and mutagenesis in Pseudomonas aeruginosa.

Differential regulation of polyphosphate genes in Pseudomonas aeruginosa.

Phosphate homeostasis is tightly regulated in bacteria. Phosphate scarcity is overcome by inducing the expression of genes associated with the scavenging of phosphate and phosphate-containing molecules, while phosphate surplus is stored in the form of polyphosphate (polyP). Regulation of the genes involved in polyP metabolism was investigated. Knockout of the most distal gene of the pstSCAB-phoU operon that encodes a Pi-transport system results in large accumulation of polyphosphate (polyP). Here, we show that the phoU mutation differentially affects the transcription of ppk and ppx, that respectively, encode a polyP kinase and a polyP exopolyphosphatase, by increasing the former and reducing the latter, further contributing the accumulation of polyP. We also show that ppk forms an operon with the upstream gene hemB and that neither ppk nor ppx positively respond to Pi starvation. Furthermore, a putative PHO-box sequence in ppx regulatory region did not show a strong affinity for the PHO response regulator PhoB, while the promoter of hemB does not carry a PHO-box sequence. Altogether, the data indicate that the main genes involved in polyP metabolism, ppk and ppx, are differentially regulated in the absence of phoU, but neither gene belongs to the PHO regulon.

Chromobacterium violaceum adaptation to low-phosphate conditions.

Chromobacterium violaceum is a free-living bacterium that inhabits low-nutrient environments such as the Amazon basin. Bacteria respond to phosphate (Pi) shortage by expressing a range of genes involved in Pi uptake and assimilation, known as the PHO regulon. Several PHO regulon genes have been annotated in the genome of C. violaceum. Here we show that C. violaceum is extremely well adapted to low-Pi conditions. Remarkably, this bacterium is able to grow in media containing only traces of Pi. The PHO regulon genes are induced upon Pi depletion, but the bacteria continued to grow under these conditions. Unlike other Proteobacteria hitherto analyzed, neither PstS nor PhoU play a role in the repression of the PHO regulon under Pi excess.

First Characterization of CTX-M-15-Producing Escherichia coli Strains Belonging to Sequence Type (ST) 410, ST224, and ST1284 from Commercial Swine in South America.

We report for the first time the isolation of CTX-M-15-producingEscherichia colistrains belonging to sequence type (ST) 410, ST224, and ST1284 in commercial swine in Brazil. TheblaCTX-M-15gene was located on F-::A9::B1 and C1::A9::B1 IncF-type plasmids, surrounded by a new genetic context comprising the IS26insertion sequence truncated with the ISEcp1element upstream ofblaCTX-M-15 These results reveal that commercial swine have become a new reservoir of CTX-M-15-producing bacteria in South America.