Impact of the ´Alforja Educativa' on Ecuadorian schoolchildren's knowledge of bacteria, antibiotics, and antibiotic resistance, a pretest-posttest study.

BACKGROUND: Widespread use of antibiotics disrupts the balance in the microbial world and promotes development and spread of antibiotic resistant bacteria. Educational initiatives are important as part of strategies to mitigate antibiotic resistance. The Alforja Educativa is an innovative educational program developed in Ecuador with the aim to teach schoolchildren about antibiotic use and antibiotic resistance. The program places antibiotic resistance within a broader frame of health, well-being, and ecological awareness, highlighting the importance to maintain balance in the microbial world. The objective of this study was to evaluate the effect of the Alforja Educativa on knowledge about bacteria, antibiotics and antibiotic resistance amongst fifth and sixth grade Ecuadorian schoolchildren. METHODS: This pretest-posttest intervention study was conducted between April and June 2017 and comprised fifth and sixth grade schoolchildren from 20 schools in Cuenca, Ecuador, recruited by purposeful sampling. The Alforja Educativa was implemented over twelve 80-minute sessions by trained university students. Schoolchildren's knowledge was assessed before and after participation in the educational program using a structured questionnaire. A mean total score, the proportion of correct responses for each individual knowledge-based question, as well as correct responses for each of the multiple-choice options of the knowledge-based questions were calculated for the pretest and posttest. RESULTS: A total of 1,257 schoolchildren participated in the Alforja Educativa program, of which 980 (78%) completed both the pretest and posttest. Overall, the mean total knowledge score increased from pretest to posttest (2.58/7.00 vs. 3.85/7.00; CI = 0.5, p < 0.001). After participation in the program, the proportion of schoolchildren that correctly identified that bacteria can be both good and bad increased from 35.0 to 84.3%. In addition, scores increased for correctly identifying the meaning of antibiotic resistance (37.4-72.0%); how to prevent antibiotic resistance (63.2-74.6%); and for identifying the meaning of self-medication (46.3-54.3%). CONCLUSION: The Alforja Educativa was effective in improving the knowledge of participating schoolchildren about concepts related to bacteria, antibiotics and antibiotic resistance. The holistic perspective taken to explain the complex relationship between humans and bacteria, as well as the effect of antibiotics on the microbial world, may help provide a foundation for more sustainable antibiotic use.

Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model.

BACKGROUND: During infection by intracellular pathogens, a highly complex interplay occurs between the infected cell trying to degrade the invader and the pathogen which actively manipulates the host cell to enable survival and proliferation. Many intracellular pathogens pose important threats to human health and major efforts have been undertaken to better understand the host-pathogen interactions that eventually determine the outcome of the infection. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. discoideum when infected with Mycobacterium marinum and Legionella pneumophila. The results were compared to available data from human macrophages. RESULTS: The majority of the transcriptional regulation triggered by the two pathogens was found to be unique for each bacterial challenge. Hallmark transcriptional signatures were identified for each infection, e.g. induction of endosomal sorting complexes required for transport (ESCRT) and autophagy genes in response to M. marinum and inhibition of genes associated with the translation machinery and energy metabolism in response to L. pneumophila. However, a common response to the pathogenic bacteria was also identified, which was not induced by non-pathogenic food bacteria. Finally, comparison with available data sets of regulation in human monocyte derived macrophages shows that the elicited response in D. discoideum is in many aspects similar to what has been observed in human immune cells in response to Mycobacterium tuberculosis and L. pneumophila. CONCLUSIONS: Our study presents high-throughput characterization of D. discoideum transcriptional response to intracellular pathogens using RNA-seq. We demonstrate that the transcriptional response is in essence distinct to each pathogen and that in many cases, the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. This indicates that host-pathogen interactions are evolutionary conserved, derived from the early interactions between free-living phagocytic cells and bacteria. Taken together, our results strengthen the use of D. discoideum as a general infection model.

Mechanisms and fitness costs of resistance to antimicrobial peptides LL-37, CNY100HL and wheat germ histones.

Antimicrobial peptides (AMPs) represent a potential new class of antimicrobial drugs with potent and broad-spectrum activities. However, knowledge about the mechanisms and rates of resistance development to AMPs and the resulting effects on fitness and cross-resistance is limited. We isolated antimicrobial peptide (AMP) resistant Salmonella typhimurium LT2 mutants by serially passaging several independent bacterial lineages in progressively increasing concentrations of LL-37, CNY100HL and Wheat Germ Histones. Significant AMP resistance developed in 15/18 independent bacterial lineages. Resistance mutations were identified by whole genome sequencing in two-component signal transduction systems (pmrB and phoP) as well as in the LPS core biosynthesis pathway (waaY, also designated rfaY). In most cases, resistance was associated with a reduced fitness, observed as a decreased growth rate, which was dependent on growth conditions and mutation type. Importantly, mutations in waaY decreased bacterial susceptibility to all tested AMPs and the mutant outcompeted the wild type parental strain at AMP concentrations below the MIC for the wild type. Our data suggests that resistance to antimicrobial peptides can develop rapidly through mechanisms that confer cross-resistance to several AMPs. Importantly, AMP-resistant mutants can have a competitive advantage over the wild type strain at AMP concentrations similar to those found near human epithelial cells. These results suggest that resistant mutants could both be selected de novo and maintained by exposure to our own natural repertoire of defence molecules.

PEG-stabilized lipid disks as carriers for amphiphilic antimicrobial peptides.

Antimicrobial peptides hold potential as a possible alternative, or complement, to conventional antibiotics but new, safe and efficient means are needed for formulation and administration of the peptides. In this study we have investigated the utility of a novel type of lipid particles, the polyethylene glycol-stabilized lipid disks, as carriers for the model peptide melittin. The structural integrity of the carrier particle when loaded with the peptide was investigated using cryo-transmission electron microscopy. Liposome leakage upon addition of the peptide-lipid disks was monitored as a means to verify the membrane lytic effect of the formulation. The susceptibility of melittin to tryptic digestion was studied and compared in the absence and presence of lipid disks. Finally, the antibacterial effect of the peptide-lipid disk formulation was compared to that of free melittin after both single and repeated exposure to Escherichia coli. The results show that melittin can redistribute from the disk into a new host membrane and that formulation in the disks does not compromise melittin's membrane permeabilizing ability. Further, the peptide was found to be fully protected against degradation when bound to the disks. Time-kill experiments revealed that all the antibacterial effect of melittin administered in free form was gone after a single exposure to E. coli. In contrast, the disk formulation showed significant cell-killing effect also upon a second exposure to bacteria, indicating an extended release of peptide from the lipid disks. These results suggest that the lipid disks constitute a new class of promising carriers for peptide antibiotics.

Activation of cryptic aminoglycoside resistance in Salmonella enterica.

Aminoglycoside resistance in bacteria can be acquired by several mechanisms, including drug modification, target alteration, reduced uptake and increased efflux. Here we demonstrate that increased resistance to the aminoglycosides streptomycin and spectinomycin in Salmonella enterica can be conferred by increased expression of an aminoglycoside adenyl transferase encoded by the cryptic, chromosomally located aadA gene. During growth in rich medium the wild-type strain was susceptible but mutations that impaired electron transport and conferred a small colony variant (SCV) phenotype or growth in glucose/glycerol minimal media resulted in activation of the aadA gene and aminoglycoside resistance. Expression of the aadA gene was positively regulated by the stringent response regulator guanosine penta/tetraphosphate ((p)ppGpp). SCV mutants carrying stop codon mutations in the hemA and ubiA genes showed a streptomycin pseudo-dependent phenotype, where growth was stimulated by streptomycin. Our data suggest that this phenotype is due to streptomycin-induced readthrough of the stop codons, a resulting increase in HemA/UbiA levels and improved electron transport and growth. Our results demonstrate that environmental and mutational activation of a cryptic resistance gene can confer clinically significant resistance and that a streptomycin-pseudo-dependent phenotype can be generated via a novel mechanism that does not involve the classical rpsL mutations.

Escape from growth restriction in small colony variants of Salmonella typhimurium by gene amplification and mutation.

Antibiotic resistance in bacteria is generally associated with fitness costs that often can be reduced by second-site compensatory mutations. Here, we examined how a protamine-resistant small colony variant of Salmonella typhimurium adapts to the growth reduction conferred by a resistance mutation in hemC (encoding a haem-biosynthesis enzyme). We show that adaptation occurs in a multi-step process where fitness is successively increased. Thus, the initial adaptive response was selection for an unstable gene amplification of the mutant hemC gene that provided a small fitness increase. Fitness was increased further by a mutation that restored HemC function in one gene copy, relaxing selection for the amplification. Subsequently, the amplification segregated back to the haploid state and even higher fitness. The end result was in most cases mutant strains with a hemC sequence different from that of the wild-type strain. These findings suggest that gene amplification facilitates adaptive evolution. A higher gene dosage increases the target size for compensatory mutations and improves fitness of the cell, thereby allowing an increase in the population size, further increasing the probability of a subsequent stable mutation. Our results provide a novel genetic basis for growth compensation in small colony variants.

The cyclotide cycloviolacin O2 from Viola odorata has potent bactericidal activity against Gram-negative bacteria.

OBJECTIVES: To determine the antibacterial activity of small cyclic plant proteins, i.e. cyclotides, and the importance of the surface exposed charged residues for activity. METHODS: Prototypic cyclotides, including the Möbius kalata B1 and the bracelet cycloviolacin O2 (cyO2), were isolated using reversed-phase HPLC. Initial activity screenings were conducted using radial diffusion assays (RDAs) and MIC assays with Salmonella enterica serovar Typhimurium LT2, Escherichia coli and Staphylococcus aureus as test strains. For the most active peptide, cyO2, time-kill kinetics was determined in sodium phosphate buffer (containing 0.03% trypticase soy broth) against several Gram-negative and Gram-positive bacterial species. Charged residues in cyO2 were chemically modified and activity was determined in time-kill assays. RESULTS: CyO2 was the most active cyclotide and efficiently inhibited the growth of S. enterica serovar Typhimurium LT2 and E. coli in RDAs and MIC assays, while the other peptides were less active. In time-kill assays, cyO2 also had bactericidal activity against the Gram-negative species Klebsiella pneumoniae and Pseudomonas aeruginosa. In contrast, none of the cyclotides had high activity against S. aureus. Chemical masking of the charged Glu and Lys residues in cyO2 caused a near total loss of activity against Salmonella, while masking Arg caused a less pronounced activity reduction. CONCLUSIONS: CyO2 is a cyclotide with potent activity against Gram-negative bacteria. The charged residues in cyO2 are all required for optimum antibacterial activity. In combination with its previously demonstrated cytotoxic activity against cancer cells and the general stability of cyclotides, cyO2 provides a promising scaffold for future drug design.

Mechanisms and physiological effects of protamine resistance in Salmonella enterica serovar Typhimurium LT2.

OBJECTIVES: Protamines are cationic peptides that exert antimicrobial activity. We have examined the evolution of bacterial resistance to protamine sulphate and the resulting effects on fitness and physiology, with the objective of increasing knowledge about mechanisms of bacterial resistance to antimicrobial peptides. METHODS: Spontaneous, protamine-resistant Salmonella enterica serovar Typhimurium (i.e. Salmonella Typhimurium) LT2 mutants were isolated on agarose plates containing protamine sulphate. Resistance mutations were identified using transposon insertions and DNA sequencing. Peptide susceptibility was determined by broth dilution tests and antibiotic susceptibility using Etests. Fitness was determined as log-phase growth rates. Growth-compensated strains were isolated by serial passage through population bottlenecks followed by visual screening for large colonies. RESULTS: Protamine-resistant mutants appeared at a rate of 2.3 x 10(-7)/cell/generation. These mutants were 2-20 times more resistant to protamine than the parental strain and less susceptible to several other antimicrobials, including colistin, gentamicin, lactoferricin and human defensin HNP-1. The resistance mutations were mapped to genes involved in haem biosynthesis and respiration, and were associated with a reduction in bacterial fitness. Some mutants could, in the absence of protamine, be evolved to higher fitness by acquiring second-site compensatory mutations. CONCLUSIONS: Spontaneous mutants resistant to protamine sulphate were readily selected in Salmonella Typhimurium LT2. These mutants were less susceptible to several other peptides and antibiotics, and had the characteristics of small colony variants, a phenotype often associated with persistent and recurrent infections that are difficult to treat and which could be a strategy for bacteria to escape the killing effects of antimicrobial peptides.

Mechanism and fitness costs of PR-39 resistance in Salmonella enterica serovar Typhimurium LT2.

PR-39 is a porcine antimicrobial peptide that kills bacteria with a mechanism that does not involve cell lysis. Here, we demonstrate that Salmonella enterica serovar Typhimurium can rapidly acquire mutations that reduce susceptibility to PR-39. Resistant mutants appeared at a rate of 0.4 x 10(-6) per cell per generation. These mutants were about four times more resistant than the wild type and showed a greatly reduced rate of killing. Genetic analysis revealed mutations in the putative transport protein SbmA as being responsible for the observed resistance. These sbmA mutants were as fit as the wild-type parental strain as measured by growth rates in culture medium and mice and by long-term survival in stationary phase. These results suggest that resistance to certain antimicrobial peptides can rapidly develop without an obvious fitness cost for the bacteria and that resistance development could become a threat to the efficacy of antimicrobial peptides if used in a clinical setting.