Search

IntroductionMurepavadin is an antimicrobial peptide (AMP) in clinical development that selectively targets Pseudomonas aeruginosa LptD and whose resistance profile remains unknown. We aimed to explore genomic modifications and consequences underlying murepavadin and/or colistin susceptibility.MethodsTo define genomic mechanisms underlying resistance, we performed two approaches: 1) a genome-wide association study (GWAS) in a P. aeruginosa clinical collection (n=496), considering >0.25 mg/L as tentative cut-off of murepavadin acquired resistance; 2) a paired genomic comparison in a subset of 5 isolates and their isogenic murepavadin-resistant mutants obtained in vitro. Lipid-A composition, immunogenicity and cathelicidin and indolicidin effects on bacterial growth were also tested in this last subset of isolates. Murepavadin MICs were determined in ΔlpxL1 and ΔlpxL2 knock-out mutants obtained from a auxotroph PAO1 derivative.ResultsGWAS revealed a missense variant (A→G p.Thr260Ala in the hisJ gene) associated with murepavadin resistance although both resistant and susceptible strains harbored it (21% and 12% respectively, OR=1.92, p=0.012 in χ² test). Among the isolate subset, murepavadin-resistant mutants with deletions in lpxL1 and lpxL2 genes showed lower abundance of hexa-acylated lipid-A (m/z 1616, 1632). 4-aminoarabinose addition was found only in colistin-resistant isolates but not in the other ones, irrespective of murepavadin susceptibility. Accordingly, ΔlpxL1 and ΔlpxL2 mutants exhibited higher murepavadin MICs than parental PAO1 auxotroph strain (2 and 4 vs 0.5 mg/L respectively). Lipopolysaccharide from murepavadin-resistant mutants triggered lower inflammatory responses in human monocytes. Those with lpxL mutations and hexa-acylated lipid-A loss also exhibited greater growth reduction when exposed to host-derived AMPs cathelicidin and indolicidin.DiscussionHigh murepavadin-resistance seems to be linked to lpxL1 and lpxL2 mutations and lower hexa-acylated lipid-A, corresponding to lower inflammatory induction and higher susceptibility to host-derived AMPs. Although GWAS identified one variant associated with the murepavadin-resistant phenotype, data revealed that there was no unique single genetic event underlying this phenotype. Our study provides insight into the mechanisms underlying murepavadin susceptibility.

Abstract The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection.

Summary: Background: Pseudomonas aeruginosa is a major cause of hospital-acquired and chronic infections, characterised by an extraordinary capacity to develop antimicrobial resistance through the selection of chromosomal mutations, leading to treatment failure. Here, we designed and tested a hybridisation-based capture system for the enrichment of genes of interest before sequencing to monitor resistant populations genomics directly from clinical samples. Methods: A panel for enrichment before sequencing of close to 200 genes related to P. aeruginosa antimicrobial resistance, multilocus sequence typing, mutability or virulence was designed, synthesised (KAPA HyperCap, Roche) and initially validated in vitro using a multidrug-resistant ST175 isolate and representative isolates from major P. aeruginosa clades. In vivo testing included ventilator associated pneumonia by MDR P. aeruginosa in ICU (3–10 sequential samples from 3 patients) and chronic respiratory infection by hypermutable P. aeruginosa in cystic fibrosis (8 sequential samples from a single patient covering a 4-year period). Results from direct sequencing with the enrichment panel were compared with those of whole genome sequencing (WGS) and phenotypic profiling of 10 isolated colonies per sample. Findings: In vitro assays confirmed the selectivity of the enrichment panel and the correct identification of the vast mutational resistome of ST175, including specific mutations even when introduced in a 1:100 proportion. In vivo performance was at least equivalent to sequencing 10 colonies per sample, including the accurate identification of the sequence types and the basal and acquired mutational resistome. To note, specific resistance mutations, such as those in ampC leading to resistance to novel β-lactams, could be traced even at frequencies of 1%. Moreover, the coselection of mutator populations and antibiotic resistance mutations, predicted in theoretical and in vitro studies, was evidenced in vivo. Interpretation: This proof-of-concept study demonstrates that resistance genomics of P. aeruginosa can be analysed directly from clinical samples, determining not only a considerable reduction in turnaround time and cost from a diagnostics perspective, but also an unprecedented potency for accurate monitoring of in vivo population dynamics in bacterial infections. Funding: Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU.

Annually, food poisoning caused by non-typhoidal Salmonella (NTS) accounts for 40 million foodborne infections worldwide. Outbreaks due to multidrug-resistant NTS strains are associated with the consumption of poultry products. We aimed to determine the proportion of NTS in ground turkey meat available at supermarkets in two locations in Chile, characterizing their serovars, virulence traits, and antimicrobial resistance profiles. Between September and December 2018, we analyzed 47 samples of packed ground turkey meat purchased at 13 supermarkets. NTS were identified by MALDI-TOF, and antimicrobial susceptibility was determined by the disk-diffusion agar method. Whole genome sequencing was performed with MiSeq (Illumina) followed by Nullarbor analysis. NTS was detected in 6 out of 47 samples (12.8 %, 95 %CI: 6.0–25.1 %), identifying S. enterica serovar Infantis (n=4 isolates), serovar Agona (n=1 isolate), and serovar Heidelberg (n=1 isolate), respectively. S. Infantis belonged to sequence type (ST)32, differentiating two clones, whereas the S. Agona was ST13 and S. Heidelberg was ST15. S. Infantis strains were multidrug antibiotic resistant, harboring blaCTX-M-65, tetA, and mutations that confer fosfomycin resistance. Serovars Agona and Heidelberg showed tetracycline (tetB) and fosfomycin resistance (fosA7). Additionally, serovar Heidelberg showed resistance to sulfamethoxazole-trimethoprim, aminoglycosides, and carried the blaCMY-2 gene. This is the first report in Chile about NTS detected in raw ground turkey meat available in supermarkets. S. Infantis and S. Agona strains showed a phylogenetic relationship with strains of the same serovars isolated from the environment in our country. This situation would impact food poisoning and the dissemination of bacterial resistance between animals, humans, and the environment.

Background/Objectives: Faecal microbiota transplantation (FMT) is considered a safe and effective therapy for recurrent Clostridioides difficile infection. It is the only current clinical indication for this technique, although numerous clinical research studies and trials propose its potential usefulness for treating other pathologies. Donor selection is a very rigorous process, based on a personal lifestyle interview and the absence of known pathogens in faeces and serum, leading to only a few volunteers finally achieving the corresponding certification. However, despite the high amount of data generated from the ongoing research studies relating microbiota and health, there is not yet a consensus defining what is a “healthy” microbiota. To date, knowledge of the composition of the microbiota is not a requirement to be a faecal donor. The aim of this work was to evaluate whether the analysis of the composition of the microbiota by massive sequencing of 16S rDNA could be useful in the selection of the faecal donors. Methods: Samples from 10 certified donors from Mikrobiomik Healthcare Company were collected and sequenced using 16S rDNA in a MiSeq (Illumina) platform. Alpha (Chao1 and Shannon indices) and beta diversity (Bray–Curtis) were performed using the bioinformatic web server Microbiome Analyst. The differences in microbial composition at the genera and phyla levels among the donors were evaluated. Results: The microbial diversity metric by alpha diversity indexes showed that most donors exhibited a similar microbial diversity and richness, whereas beta diversity by 16S rDNA sequencing revealed significant inter-donor differences, with a more stable microbial composition over time in some donors. The phyla Bacillota and Bacteroidota were predominant in all donors, while the density of other phyla, such as Actinomycota and Pseudomonota, varied among individuals. Each donor exhibited a characteristic genera distribution pattern; however, it was possible to define a microbiome core consisting of the genera Agathobacter, Eubacterium, Bacteroides, Clostridia UCG-014 and Akkermansia. Conclusions: The results suggest that donor certification does not need to rely exclusively on their microbiota composition, as it is unique to each donor. While one donor showed greater microbial diversity and richness, clear criteria for microbial normality and health have yet to be established. Therefore, donor certification should focus more on clinical and lifestyle aspects.

Antimicrobial resistance (AMR) surveillance in fecal Escherichia coli isolates from wildlife is crucial for monitoring the spread of this microorganism in the environment and for developing effective AMR control strategies. Wildlife can act as carriers of AMR bacteria and spread them to other wildlife, domestic animals, and humans; thus, they have public health implications. A total of 128 Escherichia coli isolates were obtained from 66 of 217 fecal samples obtained from different wild animals using media without antibiotic supplementation. Antibiograms were performed for 17 antibiotics to determine the phenotypic resistance profile in these isolates. Extended-spectrum β-lactamase (ESBL) production was tested using the double-disc synergy test, and 29 E. coli strains were selected for whole genome sequencing. In total, 22.1% of the wild animals tested carried multidrug-resistant E. coli isolates, and 0.93% (2/217) of these wild animals carried E. coli isolates with ESBL-encoding genes (blaCTX-M-65, blaCTX-M-55, and blaEC-1982). The E. coli isolates showed the highest resistance rates to ampicillin and were fully susceptible to amikacin, meropenem, ertapenem, and imipenem. Multiple resistance and virulence genes were detected, as well as different plasmids. The relatively high frequency of multidrug-resistant E. coli isolates in wildlife, with some of them being ESBL producers, raises some concern regarding the potential transmission of antibiotic-resistant bacteria among these animals. Gaining insights into antibiotic resistance patterns in wildlife can be vital in shaping conservation initiatives and developing effective strategies for responsible antibiotic use.

ABSTRACTSerratia spp. is a well-recognized pathogen in neonates; however, limited data are available in adults. We studied microbiological and clinical characteristics of Serratia spp. causing bloodstream infections (BSI) in our institution (January 2005–July 2020). Overall, 141 BSI episodes affecting 139 patients were identified and medical records reviewed. Antimicrobial susceptibility was recovered from our informatics system and 118 isolates from 116 patients were available for further microbiological studies. Whole genome sequencing (WGS) was completed in 107 isolates. Incidence of Serratia BSI was 0.3/1000 overall admissions (range 0.12–0.60), with maximum prevalence (27 episodes, 19.1%) during 2017–2018. Relevant patients’ clinical characteristics were 71.9% ≥60 years (n = 100), with high comorbidity rates (49%, ≥2), 23 (74.2%) of them died within 1 month of the BSI episode. WGS identified all isolates as Serratia marcescens when Kraken bioinformatics taxonomic tool was used despite some which were identified as Serratia nematodiphila (32/118) or Serratia ureilytica (5/118) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nevertheless, when using MASH distance, Serratia nevei (63/107), S. ureilytica (38/107), and S. marcescens (6/107) were assigned. Carbapenemase (blaVIM-1) and extended-spectrum β-lactases (ESBL) (blaSHV-12) genes were found in seven and three isolates, respectively, one of them expressing both genes. The worldwide-disseminated IncL/M scaffold plasmid was identified in six VIM producers. Four genotypes were established based on their virulence factors and resistome. Serratia spp. emerged as a relevant nosocomial pathogen causing BSI in elderly patients in our hospital, particularly in recent years with a remarkable increase in antibiotic resistance. ESBL and carbapenemases production related to plasmid dissemination are particularly noteworthy.IMPORTANCESerratia spp. is the third most frequent pathogen involved in outbreaks at neonatal facilities and is primarily associated with bacteremia episodes. In this study, we characterized all causing bloodstream infection (BSI) in patients admitted to our hospital during a 16-year period (2005–2020). Despite having no neonatal intensive care unit in our hospital, this study revealed that Serratia spp. is a relevant pathogen causing BSI in elderly patients with high comorbidity rates. A significant increase of antimicrobial resistance was detected over time, particularly in 2020 and coinciding with the coronavirus disease (COVID-19) pandemic and nosocomial spread of multidrug-resistant Serratia spp. isolates. extended-spectrum β-lactases and carbapenemases genes associated with plasmid dissemination, typically detected in other Enterobacterales species, were also identified, reinforcing the role of Serratia spp. in the antimicrobial resistance landscape. Additionally, this work highlights the need to reclassify the species of Serratia, since discrepancies were observed in the identification when using different tools.

Acute myocardial infarction (AMI) is associated with systemic inflammatory processes and metabolic alterations. Microbial-derived metabolites, such as short-chain fatty acids and trimethylamine N-oxide (TMAO), have emerged in recent years as key players in the modulation of inflammation, with potential implications for cardiovascular diseases. We performed a prospective observational study that monitored the serological concentration of bacterial metabolites in 45 young patients (

Abstract This work aimed to evaluate the predatory activity of Bdellovibrio bacteriovorus 109J on clinical isolates of Pseudomonas aeruginosa selected from well-characterized collections of cystic fibrosis (CF) lung colonization (n = 30) and bloodstream infections (BSI) (n = 48) including strains selected by genetic lineage (frequent and rare sequence types), antibiotic resistance phenotype (susceptible and multidrug-resistant isolates), and colony phenotype (mucoid and non-mucoid isolates). The intraspecies predation range (I-PR) was defined as the proportion of susceptible strains within the entire collection. In contrast, the predation efficiency (PE) is the ratio of viable prey cells remaining after predation compared to the initial inoculum. I-PR was significantly higher for CF (67%) than for BSI P. aeruginosa isolates (35%) probably related to an environmental origin of CF strains whereas invasive strains are more adapted to humans. I-PR correlation with bacterial features such as mucoid morphotype, genetic background, or antibiotic susceptibility profile was not detected. To test the possibility of increasing I-PR of BSI isolates, a polyhydroxyalkanoate depolymerase deficient B. bacteriovorus bd2637 mutant was used. Global median I-PR and PE values remained constant for both predators, but 31.2% of 109J-resistant isolates were susceptible to the mutant, and 22.9% of 109J-susceptible isolates showed resistance to predation by the mutant, pointing to a predator–prey specificity process. The potential use of predators in the clinical setting should be based on the determination of the I-PR for each species, and the PE of each particular target strain.

The acquisition and expression of antibiotic resistance implies changes in bacterial cell physiology, imposing fitness costs. Many human opportunistic pathogenic bacteria, such as those causing urinary tract or bloodstream infections, colonize the gut. In this opinionated review, we will examine the various types of stress that these bacteria might suffer during their intestinal stay. These stresses, and their compensatory responses, probably have a fitness cost, which might be additive to the cost of expressing antibiotic resistance. Such an effect could result in a disadvantage relative to antibiotic susceptible populations that might replace the resistant ones. The opinion proposed in this paper is that the effect of these combinations of fitness costs should be tested in antibiotic resistant bacteria with susceptible ones as controls. This testing might provide opportunities to increase the bacterial gut stress boosting physiological biomolecules or using dietary interventions. This approach to reduce the burden of antibiotic-resistant populations certainly must be answered empirically. In the end, the battle against antibiotic resistance should be won by antibiotic-susceptible organisms. Let us help them prevail.

The indiscriminate use of antibiotics has contributed to the dissemination of multiresistant bacteria, which represents a public health concern. The aim of this work was to characterize 27 coagulase-negative staphylococci (CoNS) isolated from eight wild Northeast Atlantic hakes (Merluccius merluccius, L.) and taxonomically identified as Staphylococcus epidermidis (n = 16), Staphylococcus saprophyticus (n = 4), Staphylococcus hominis (n = 3), Staphylococcus pasteuri (n = 2), Staphylococcus edaphicus (n = 1), and Staphylococcus capitis (n = 1). Biofilm formation was evaluated with a microtiter assay, antibiotic susceptibility testing was performed using the disk diffusion method, and antibiotic resistance and virulence determinants were detected by PCR. Our results showed that all staphylococci produced biofilms and that 92.6% of the isolates were resistant to at least one antibiotic, mainly penicillin (88.8%), fusidic acid (40.7%), and erythromycin (37%). The penicillin resistance gene (blaZ) was detected in 66.6% (18) of the isolates, of which 10 also carried resistance genes to macrolides and lincosamides (mphC, msr(A/B), lnuA, or vgaA), 4 to fusidic acid (fusB), and 3 to trimethoprim-sulfamethoxazole (dfrA). At least one virulence gene (scn, hla, SCCmecIII, and/or SCCmecV) was detected in 48% of the isolates. This study suggests that wild European hake destined for human consumption could act as a vector of CoNS carrying antibiotic resistance genes and/or virulence factors.

BackgroundDysbiosis and mucin depletion are related with intestinal barrier dysfunction and seems to be an early pathophysiological event in inflammatory bowel disease (IBD). The objective of this work is to study these parameters in the natural history of colitis in IL-10 deficient mice (IL-10−/−).MethodsWild type (WT) and IL-10−/−. mice were followed until sacrifice at 3, 5, 10, 20, 57, and 70 weeks. Body weight, colonic weight/length ratio and in vivo intestinal permeability were registered. Expression of inflammatory and adhesion molecules in the colon was explored by qPCR as Mucin-2 (MUC-2) and molecules involved in goblet cell maturation Interleukin-18 (IL-18) and WAP Four-Disulfide Core Domain 2 (WFDC2), the endoplasmic reticulum stress markers X-box-binding protein (Xbp-1) and Reticulon-4B (RTN-4B). Bacterial composition in feces and colonic mucosa was determined by massive sequencing of the V3–V4 regions of 16S rDNA gene.ResultsIL-10-/- mice showed histological inflammation at weeks 20 and 57, but most notably the intestinal permeability was significantly higher from week 10. Concordantly, the number of goblet cells and expression of MUC-2, IL-18, WFDC2 and Xbp-1 were significantly lower in KO from week 10. Nevertheless, no significant differences were found in the mRNA expression of MUC-2 or Xbp-1 between both groups—derived colon organoids. Significant bacterial differences began at week 5, being the Akkermansia deficiency in KO the most relevant result.ConclusionGut microbiota alterations and mucin depletion are associated with early intestinal barrier dysfunction and precede overt gut inflammation in this animal model of IBD.

The aim of this study was to monitor the impact of a preoperative low-calorie diet and bariatric surgery on the bacterial gut microbiota composition and functionality in severe obesity and to compare sleeve gastrectomy (SG) versus Roux-en-Y gastric bypass (RYGB). The study also aimed to incorporate big data analysis for the omics results and machine learning by a Lasso-based analysis to detect the potential markers for excess weight loss. Forty patients who underwent bariatric surgery were recruited (14 underwent SG, and 26 underwent RYGB). Each participant contributed 4 fecal samples (baseline, post-diet, 1 month after surgery and 3 months after surgery). The bacterial composition was determined by 16S rDNA massive sequencing using MiSeq (Illumina). Metabolic signatures associated to fecal concentrations of short-chain fatty acids, amino acids, biogenic amines, gamma-aminobutyric acid and ammonium were determined by gas and liquid chromatography. Orange 3 software was employed to correlate the variables, and a Lasso analysis was employed to predict the weight loss at the baseline samples. A correlation between Bacillota (formerly Firmicutes) abundance and excess weight was observed only for the highest body mass indexes. The low-calorie diet had little impact on composition and targeted metabolic activity. RYGB had a deeper impact on bacterial composition and putrefactive metabolism than SG, although the excess weight loss was comparable in the two groups. Significantly higher ammonium concentrations were detected in the feces of the RYGB group. We detected individual signatures of composition and functionality, rather than a gut microbiota characteristic of severe obesity, with opposing tendencies for almost all measured variables in the two surgical approaches. The gut microbiota of the baseline samples was not useful for predicting excess weight loss after the bariatric process.

ABSTRACT Newborn screening for cystic fibrosis (CF) can identify affected but asymptomatic infants. The selection of omic technique for gut microbiota study is crucial due to both the small amount of feces available and the low microorganism load. Our aims were to compare the agreement between 16S rRNA amplicon sequencing and metaproteomics by a robust statistical analysis, including both presence and abundance of taxa, to describe the sequential establishment of the gut microbiota during the first year of life in a small size sample (8 infants and 28 fecal samples). The taxonomic assignations by the two techniques were similar, whereas certain discrepancies were observed in the abundance detection, mostly the lower predicted relative abundance of Bifidobacterium and the higher predicted relative abundance of certain Firmicutes and Proteobacteria by amplicon sequencing. During the first months of life, the CF gut microbiota is characterized by a significant enrichment of Ruminococcus gnavus, the expression of certain virulent bacterial traits, and the detection of human inflammation-related proteins. Metaproteomics provides information on composition and functionality, as well as data on host-microbiome interactions. Its strength is the identification and quantification of Actinobacteria and certain classes of Firmicutes, but alpha diversity indices are not comparable to those of amplicon sequencing. Both techniques detected an aberrant microbiota in our small cohort of infants with CF during their first year of life, dominated by the enrichment of R. gnavus within a human inflammatory environment. IMPORTANCE In recent years, some techniques have been incorporated for the study of microbial ecosystems, being 16S rRNA gene sequencing being the most widely used. Metaproteomics provides the advantage of identifying the interaction between microorganisms and human cells, but the available databases are less extensive as well as imprecise. Few studies compare the statistical differences between the two techniques to define the composition of an ecosystem. Our work shows that the two methods are comparable in terms of microorganism identification but provide different results in alpha diversity analysis. On the other hand, we have studied newborns with cystic fibrosis, for whom we have described the establishment of an intestinal ecosystem marked by the inflammatory response of the host and the enrichment of Ruminococcus gnavus.

ABSTRACT: Objective: Men have been considered to have a higher incidence of infectious diseases, with controversy over the possibility that sex could influence the prognosis of the infection. This study aimed to explore this assumption in patients admitted to the intensive care unit (ICU) with septic bacteremia. Methods: A retrospective analysis (2006-2017) of septic patients with microbiologically confirmed bacteremia (n=440) was performed. Risk of ICU and in-hospital mortality in males versus females was compared by univariate analysis and a propensity score analysis integrating their clinical characteristics. Results: Sepsis more frequently occurred in males (80.2% vs 76.1%) as well as in-hospital (48.0% vs 41.3%) and ICU (39.9% vs 36.5%) mortality. Univariate analyses showed that males had a higher Charlson comorbidity index and worse McCabe prognostic score. However, the propensity score in 296 matched patients demonstrated that females had higher risk of both ICU (OR 1.39; 95% CI 0.89-2.19) and in-hospital mortality (OR 1.18; 95% CI 0.77-1.83), but without statistical significance. Conclusion: Males with sepsis had worse clinical characteristics when admitted to the ICU, but sex had no influence on mortality. These data contribute to helping reduce the sex-dependent gap present in healthcare provision.

A proper NADH/NAD + balance allows for the flow of metabolic and catabolic activities determining cellular growth. In Escherichia coli, more than 80 NAD + dependent enzymes are involved in all major metabolic pathways, including the post-transcriptional build-up of thiazole and oxazole rings from small linear peptides, which is a critical step for the antibiotic activity of some microcins. In recent years, NAD metabolism boosting drugs have been explored, mostly precursors of NAD + synthesis in human cells, with beneficial effects on the aging process and in preventing oncological and neurological diseases. These compounds also enhance NAD + metabolism in the human microbiota, which contributes to these beneficial effects. On the other hand, inhibition of NAD + metabolism has been proposed as a therapeutic approach to reduce the growth and propagation of tumor cells and mitigating inflammatory bowel diseases; in this case, the activity of the microbiota might mitigate therapeutic efficacy. Antibiotics, which reduce the effect of microbiota, should synergize with NAD + metabolism inhibitors, but these drugs might increase the proportion of antibiotic persistent populations. Conversely, antibiotics might have a stronger killing effect on bacteria with active NAD + production and reduce the cooperation of NAD + producing bacteria with tumoral cells. The use of NADH/NAD + modulators should take into consideration the use of antibiotics and the population structure of the microbiota.

The epidermis constitutes a continuous external layer covering the body, offering protection against bacteria, the most abundant living organisms that come into contact with this barrier. The epidermis is heavily colonized by commensal bacterial organisms that help protect against pathogenic bacteria. The highly regulated and dynamic interaction between the epidermis and commensals involves the host’s production of nutritional factors promoting bacterial growth together to chemical and immunological bacterial inhibitors. Signal trafficking ensures the system’s homeostasis; conditions that favor colonization by pathogens frequently foster commensal growth, thereby increasing the bacterial population size and inducing the skin’s antibacterial response, eliminating the pathogens and re-establishing the normal density of commensals. The microecological conditions of the epidermis favors Gram-positive organisms and are unsuitable for long-term Gram-negative colonization. However, the epidermis acts as the most important host-to-host transmission platform for bacteria, including those that colonize human mucous membranes. Bacteria are frequently shared by relatives, partners, and coworkers. The epidermal bacterial transmission platform of healthcare workers and visitors can contaminate hospitalized patients, eventually contributing to cross-infections. Epidermal transmission occurs mostly via the hands and particularly through fingers. The three-dimensional physical structure of the epidermis, particularly the fingertips, which have frictional ridges, multiplies the possibilities for bacterial adhesion and release. Research into the biology of bacterial transmission via the hands is still in its infancy; however, tribology, the science of interacting surfaces in relative motion, including friction, wear and lubrication, will certainly be an important part of it. Experiments on finger-to-finger transmission of microorganisms have shown significant interindividual differences in the ability to transmit microorganisms, presumably due to genetics, age, sex, and the gland density, which determines the physical, chemical, adhesive, nutritional, and immunological status of the epidermal surface. These studies are needed to optimize interventions and strategies for preventing the hand transmission of microorganisms.

Objective To assess the role of sex as an independent prognostic factor for mortality in patients with sepsis admitted to intensive care units (ICUs).Design Systematic review and meta-analysis.Data sources MEDLINE, Embase, Web of Science, ClinicalTrials.gov and the WHO Clinical Trials Registry from inception to 17 July 2020.Study selection Studies evaluating independent associations between sex and mortality in critically ill adults with sepsis controlling for at least one of five core covariate domains prespecified following a literature search and consensus among experts.Data extraction and synthesis Two authors independently extracted and assessed the risk of bias using Quality In Prognosis Studies tool. Meta-analysis was performed by pooling adjusted estimates. The Grades of Recommendations, Assessment, Development and Evaluation approach was used to rate the certainty of evidence.Results From 14 304 records, 13 studies (80 520 participants) were included. Meta-analysis did not find sex-based differences in all-cause hospital mortality (OR 1.02, 95% CI 0.79 to 1.32; very low-certainty evidence) and all-cause ICU mortality (OR 1.19, 95% CI 0.79 to 1.78; very low-certainty evidence). However, females presented higher 28-day all-cause mortality (OR 1.18, 95% CI 1.05 to 1.32; very low-certainty evidence) and lower 1-year all-cause mortality (OR 0.83, 95% CI 0.68 to 0.98; low-certainty evidence). There was a moderate risk of bias in the domain adjustment for other prognostic factors in six studies, and the certainty of evidence was further affected by inconsistency and imprecision.Conclusion The prognostic independent effect of sex on all-cause hospital mortality, 28-day all-cause mortality and all-cause ICU mortality for critically ill adults with sepsis was uncertain. Female sex may be associated with decreased 1-year all-cause mortality.PROSPERO registration number CRD42019145054.

Abstract Background Nosocomial sepsis is the main problem that preterms have to face during their stay at neonatal intensive care units (NICU). Serratia marcescens is an emerging cause of preterm sepsis but its epidemiology is still largely unknown. Consequently, the aims of this study were to know the rate of preterms colonized by S. marcescens during their stay at the NICU and the characteristics and evolution of the S. marcescens population, including the susceptibility to clinically relevant antibiotics. Methods Twenty-six preterm infants born with a gestational age ≤ 32 weeks and/or weigh ≤1500 g were included in the study. Samples of meconium and feces (n = 92) were collected during their first month of life of the infants, together with feeding samples after their pass through enteral feeding tubes (n = 37). Samples were inoculated on MacConkey agar plates. The isolates identified as S. marcescens were genotyped using RAPD and PFGE; and antibiotics susceptibility was performed in a Vitek 2 system. Results A total of 179 S. marcescens isolates were obtained from the samples. PFGE profiling and cluster analysis allowed the classification of the isolates into 7 different S. marcescens clones. PFGE patterns 1 and 3 were the dominant strains in the fecal samples colonizing 31 and 35% of the infants, respectively. Those isolates causing bacteremia in two infants clustered in PFGE pattern 3. Conclusion S. marcescens is a bacterial species closely associated to the NICU environment. It can be frequently isolated from preterm’s feces although only some genetic lineages seem to be associated to sepsis. Enteral feeding tubes act as important reservoirs to keep the S. marcescens population in the NICU. Trial registration The local ethic committee approved this trial with the reference 09/157.

Intestinal microbiota seems to play a key role in obesity. The impact of the composition and/or functionality of the obesity-associated microbiota have yet to be fully characterized. This work assessed the significance of the taxonomic composition and/or metabolic activity of obese- microbiota by massive 16S rRNA gene sequencing of the fecal microbiome of obese and normoweight individuals. The obese metabolic activity was also assessed by in vitro incubation of obese and normoweight microbiotas in nutritive mediums with different energy content. We found that the microbiome richness and diversity of the two groups did not differ significantly, except for Chao1 index, significantly higher in normoweight individuals. At phylum level, neither the abundance of Firmicutes or Bacteroidetes nor their ratio was associated with the body mass index. Besides, the relative proportions in Collinsella, Clostridium XIVa, and Catenibacterium were significantly enriched in obese participants, while Alistipes, Clostridium sensu stricto, Romboutsia, and Oscillibacter were significantly diminished. In regard to metabolic activity, short-chain fatty acids content was significant higher in obese individuals, with acetate being the most abundant followed by propionate and butyrate. Acetate and butyrate production was also higher when incubating obese microbiota in mediums mimicking diets with different energy content; interestingly, a reduced capability of propionate production was associated to the obese microbiome. In spite of the large interindividual variability, the obese phenotype seems to be defined more by the abundance and/or the absence of distinct communities of microorganism rather than by the presence of a specific population.

Fecal microbiota transplantation (FMT) is an effective procedure against Clostridioides difficile infection (CDI), with promising but still suboptimal performance in other diseases, such as ulcerative colitis (UC). The recipient’s mucosal immune response against the donor’s microbiota could be relevant factor in the effectiveness of FMT. Our aim was to design and validate an individualized immune-based test to optimize the fecal donor selection for FMT. First, we performed an in vitro validation of the test by co-culturing lymphocytes obtained from the small intestine mucosa of organ donor cadavers (n=7) and microbe-associated molecular patterns (MAMPs) obtained from the feces of 19 healthy donors. The inflammatory response was determined by interleukin supernatant quantification using the Cytometric Bead Array kit (B&D). We then conducted a clinical pilot study with 4 patients with UC using immunocompetent cells extracted from rectal biopsies and MAMPs from 3 donor candidates. We employed the test results to guide donor selection for FMT, which was performed by colonoscopy followed by 4 booster instillations by enema in the following month. The microbiome engraftment was assessed by 16S rDNA massive sequencing in feces, and the patients were clinically followed-up for 16 weeks. The results demonstrated that IL-6, IL-8, and IL-1ß were the most variable markers, although we observed a general tolerance to the microbial insults. Clinical and colonoscopy remission of the patients with UC was not achieved after 16 weeks, although FMT provoked enrichment of the Bacteroidota phylum and Prevotella genus, with a decrease in the Actinobacteriota phylum and Agathobacter genus. The most relevant result was the lack of Akkermansia engraftment in UC. In summary, the clinical success of FMT in patients with UC appears not to be influenced by donor selection based on the explored recipient’s local immunological response to FMT, suggesting that this approach would not be valid for FMT fecal donor optimization in such patients.

Background Testing used in screening, diagnosis and follow-up of COVID-19 has been a subject of debate. Several organisations have developed formal advice about testing for COVID-19 to assist in the control of the disease. We collated, delineated and appraised current worldwide recommendations about the role and applications of tests to control SARS-CoV-2/COVID-19.Methods We searched for documents providing recommendations for COVID-19 testing in PubMed, EMBASE, LILACS, the Coronavirus Open Access Project living evidence database and relevant websites such as TRIP database, ECRI Guidelines Trust, the GIN database, from inception to 21 September 2020. Two reviewers applied the eligibility criteria to potentially relevant citations without language or geographical restrictions. We extracted data in duplicate, including assessment of methodological quality using the Appraisal of Guidelines for Research and Evaluation-II tool.Results We included 47 relevant documents and 327 recommendations about testing. Regarding the quality of the documents, we found that the domains with the lowest scores were ‘Editorial independence’ (Median=4%) and ‘Applicability’ (Median=6%). Only six documents obtained at least 50% score for the ‘Rigour of development’ domain. An important number of recommendations focused on the diagnosis of suspected cases (48%) and deisolation measures (11%). The most frequently recommended test was the reverse transcription-PCR (RT-PCR) assay (87 recommendations) and the chest CT (38 recommendations). There were 22 areas of agreement among guidance developers, including the use of RT-PCR for SARS-Cov-2 confirmation, the limited role of bronchoscopy, the use chest CT and chest X-rays for grading severity and the co-assessment for other respiratory pathogens.Conclusion This first scoping review of recommendations for COVID-19 testing showed many limitations in the methodological quality of included guidance documents that could affect the confidence of clinicians in their implementation. Future guidance documents should incorporate a minimum set of key methodological characteristics to enhance their applicability for decision making.

The first documented nosocomial outbreak caused by Serratia marcescens in Spain occurred in 1969 at the neonatal intensive care unit (NICU) of the tertiary La Paz Children’s Hospital in Madrid, Spain, and based on the available phenotyping techniques at this time, it was considered as a monoclonal outbreak. Only 47 years later, another S. marcescens outbreak of an equivalent dimension occurred at the same NICU. The aim of the present study was to study isolates from these historical and contemporary outbreaks by phenotypic analysis and whole-genome sequencing techniques and to position these strains along with 444 publicly available S. marcescens genomes, separately comparing core genome and accessory genome contents. Clades inferred by both approaches showed high correlation, indicating that core and accessory genomes seem to evolve in the same manner for S. marcescens. Nine S. marcescens clusters were identified, and isolates were grouped in two of them according to sampling year. One exception was isolate 13F-69, the most genetically distant strain, located in a different cluster. Categorical functions in the annotated accessory genes of both collections were preserved among all isolates. No significant differences in frequency of insertion sequences in historical (0.18–0.20)—excluding the outlier strain—versus contemporary isolates (0.11–0.19) were found despite the expected resting effect. The most dissimilar isolate, 13F-69, contains a highly preserved plasmid previously described in Bordetella bronchiseptica. This strain exhibited a few antibiotic resistance genes not resulting in a resistant phenotype, suggesting the value of gene down expression in adaptation to long-term starvation.

Microbes play an important role in the pathogenesis of chronic lung diseases, such as chronic obstructive pulmonary disease, cystic fibrosis, non-cystic fibrosis bronchiectasis, and asthma. While the role of bacterial pathogens has been extensively studied, the contribution of fungal species to the pathogenesis of chronic lung diseases is much less understood. The recent introduction of next-generation sequencing techniques has revealed the existence of complex microbial lung communities in healthy individuals and patients with chronic respiratory disorders, with fungi being an important part of these communities’ structure (mycobiome). There is growing evidence that the components of the lung mycobiome influence the clinical course of chronic respiratory diseases, not only by direct pathogenesis but also by interacting with bacterial species and with the host’s physiology. In this article, we review the current knowledge on the role of fungi in chronic respiratory diseases, which was obtained by conventional culture and next-generation sequencing, highlighting the limitations of both techniques and exploring future research areas.

Probiotics are a viable alternative to traditional chemotherapy agents to control infectious diseases in aquaculture. In this regard, Lactococcus lactis subsp. cremoris WA2-67 has previously demonstrated several probiotic features, such as a strong antimicrobial activity against ichthyopathogens, survival in freshwater, resistance to fish bile and low pH, and hydrophobicity. The aim of this manuscript is an in silico analysis of the whole-genome sequence (WGS) of this strain to gain deeper insights into its probiotic properties and their genetic basis. Genomic DNA was purified, and libraries prepared for Illumina sequencing. After trimming and assembly, resulting contigs were subjected to bioinformatic analyses. The draft genome of L. cremoris WA2-67 consists of 30 contigs (2,573,139 bp), and a total number of 2493 coding DNA sequences (CDSs). Via in silico analysis, the bacteriocinogenic genetic clusters encoding the lantibiotic nisin Z (NisZ) and two new bacteriocins were identified, in addition to several probiotic traits, such as the production of vitamins, amino acids, adhesion/aggregation, and stress resistance factors, as well as the absence of transferable antibiotic resistance determinants and genes encoding detrimental enzymatic activities and virulence factors. These results unveil diverse beneficial properties that support the use of L. cremoris WA2-67 as a probiotic for aquaculture.

Klebsiella spp. is a relevant pathogen that can present acquired resistance to almost all available antibiotics, thus representing a serious threat for public health. While most studies have been focused on isolates causing community-acquired and nosocomial infections, little is known about the commensal isolates colonizing healthy subjects. We describe the molecular identification and the phenotypic characterization of commensal Klebsiella spp. from breast milk of healthy women and faeces from healthy breast-fed infants, which were compared with isolates from community-acquired infections and from a nosocomial NICU outbreak. The phylogenetic analysis of a 454-bp sequence of the rpoB gene was useful for species identification (K. pneumoniae, K. variicola, K. quasipneumoniae, K. oxytoca, K. grimontii, K. michiganensis, Raoultella planticola and R. ornithinolytica), previously misidentified as K. pneumoniae or K. oxytoca by biochemical methods. Globally, we report that commensal strains present virulence traits (virulence genes, siderophores and biofilms) comparable to community-acquired and NICU-infective isolates, thus suggesting that the human microbiota could constitute a reservoir for infection. Isolates causing NICU outbreak were multi-drug resistant (MDR) and ESBLs producers, although an imipenem-resistant commensal MDR K. quasipneumoniae isolate was also found. A commensal K. pneumoniae strain showed a potent bacteriocin-like inhibitory activity against MDR Klebsiella isolates, thus highlighting the potential role of commensal Klebsiella spp. in health and disease.

The aim of the present work was to compare the impact on the gut microbiota composition and in the systemic inflammation of two types of breads denominated as industrial and celta with different composition (wheat flour vs. complex flour and seeds) and fermentation process (2 h vs. 24 h). Faecal and blood samples of 10 mice were collected at: (1) - basal, (2) - after 21 days of industrial bread intake and (3) - after another 21 days of celta bread exposure. Significant differences in the gut microbiota composition were detected after the consumption of both breads, whereas the celta bread increased the concentration of Akkermansia and Mucispirillum, the industrial bread favour the Bacteroidetes proliferation and additionally systemic inflammation. In conclusion, the more consumed industrial processed bread might provoke systemic inflammation, probably in relation with its composition and manufacture process.

Objective: Information about antibiotic prescription patterns for cystic fibrosis (CF) patients and, specifically, about inhaled treatment strategies for their management is lacking in Spain due to the absence of a national patient registry. In this study we present data about antibiotic prescription in the Spanish CF context that were obtained in a multicenter study, being inhaled treatment strategies the special focus of this work.Methods: Twenty-four specialized CF units (12 adult, 12 pediatric) from 17 tertiary- care hospitals covering all Spanish Autonomous Communities provided sputa and clinical data from 15 consecutive patients. Data about antibiotic and non-antibiotic therapies prescribed to these patients during the year prior inclusion (2013) were retrospectively collected.Results: The multicenter study included 341 CF patients from all age groups and clinical status. The prevalence of oral, inhaled and intravenous therapies was 89% (n = 302), 80% (n = 273) and 31% (n = 105), respectively. The most prevalent oral agents were ciprofloxacin (n = 177, 59%), cotrimoxazole (n = 109, 36%) and amoxicillin-clavulanate (n = 99, 33%), whereas ceftazidime (n = 53, 50%), tobramycin (n = 43, 41%) and meropenem (n = 41, 49%) were the most prevalent intravenous ones. Two or more different inhaled antibiotics were administered to 67 patients (24%), 51 of them receiving 2 drugs continuously in alternating schemes. Nebulization of intravenous specific antibiotics was common (n = 39) and, in some cases, was used for maintenance purposes.Conclusions: These results show that the treatment of CF patients is evolving more rapidly than clinical consensus guidelines. Clinical trials evaluating new specific inhaled combinations and new alternative treatment regimes of the existing ones are needed.

BackgroundA false-negative case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is defined as a person with suspected infection and an initial negative result by reverse transcription-polymerase chain reaction (RT-PCR) test, with a positive result on a subsequent test. False-negative cases have important implications for isolation and risk of transmission of infected people and for the management of coronavirus disease 2019 (COVID-19). We aimed to review and critically appraise evidence about the rate of RT-PCR false-negatives at initial testing for COVID-19.MethodsWe searched MEDLINE, EMBASE, LILACS, as well as COVID-19 repositories, including the EPPI-Centre living systematic map of evidence about COVID-19 and the Coronavirus Open Access Project living evidence database. Two authors independently screened and selected studies according to the eligibility criteria and collected data from the included studies. The risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. We calculated the proportion of false-negative test results using a multilevel mixed-effect logistic regression model. The certainty of the evidence about false-negative cases was rated using the GRADE approach for tests and strategies. All information in this article is current up to July 17, 2020.ResultsWe included 34 studies enrolling 12,057 COVID-19 confirmed cases. All studies were affected by several risks of bias and applicability concerns. The pooled estimate of false-negative proportion was highly affected by unexplained heterogeneity (tau-squared = 1.39; 90% prediction interval from 0.02 to 0.54). The certainty of the evidence was judged as very low due to the risk of bias, indirectness, and inconsistency issues.ConclusionsThere is substantial and largely unexplained heterogeneity in the proportion of false-negative RT-PCR results. The collected evidence has several limitations, including risk of bias issues, high heterogeneity, and concerns about its applicability. Nonetheless, our findings reinforce the need for repeated testing in patients with suspicion of SARS-Cov-2 infection given that up to 54% of COVID-19 patients may have an initial false-negative RT-PCR (very low certainty of evidence).Systematic review registrationProtocol available on the OSF website: https://tinyurl.com/vvbgqya.

Streptococcus pneumoniae infections are one of the major causes of morbility and mortality worldwide. Although vaccination and antibiotherapy constitute fundamental and complementary strategies against pneumococcal infections, they present some limitations including the increase in non-vaccine serotypes and the emergence of multidrug-resistances, respectively. Ribosomally-synthesized antimicrobial peptides (i.e. bacteriocins) produced by Lactic Acid Bacteria (LAB) may represent an alternative or complementary strategy to antibiotics for the control of pneumococal infections. We tested the antimicrobial activity of 37 bacteriocinogenic LAB, isolated from food and other sources, against clinical S. pneumoniae strains. Streptococcus infantarius subsp. infantarius LP90, isolated from Venezuelan water-buffalo milk, was selected because of its broad and strong anti-pneumococcal spectrum. The in vitro safety assessment of S. infantarius LP90 revealed that it may be considered avirulent. The analysis of a 19,539-bp cluster showed the presence of 29 putative open reading frames (ORFs), including the genes encoding 8 new class II-bacteriocins, as well as the proteins involved in their secretion, immunity and regulation. Transcriptional analyses evidenced that the induction factor (IF) structural gene, the bacteriocin/IF transporter genes, the bacteriocin structural genes and most of the bacteriocin immunity genes were transcribed. MALDI-TOF analyses of peptides purified using different multichromatographic procedures revealed that the dairy strain S. infantarius LP90 produces at least 6 bacteriocins, including infantaricin A1, a novel anti-pneumococcal two-peptide bacteriocin.

Staphylococcus epidermidis has emerged as the leading agent causing neonatal late-onset sepsis in preterm neonates; although the severity of the episodes caused by this species is often underestimated, it might exert relevant short- and long-term detrimental effects on neonatal outcomes. In this context, the objective of this study was to characterize a collection of S. epidermidis strains obtained from meconium and feces of preterm infants, and to assess the potential role of the enteral feeding tubes as potential reservoirs for this microorganism. A total of 26 preterm infants were enrolled in the study. Meconium and fecal samples were collected weekly during their first month of life (n = 92). Feeding samples were collected after their pass through the enteral feeding tubes (n = 84). S. epidermidis was present in the fecal samples of all the infants in, at least, one sampling time at concentrations ranging from 6.5 to 7.8 log10 CFU/g. Initially, 344 isolates were obtained and pulsed-field gel electrophoresis (PFGE) profiling allowed the reduction of the collection to 101 strains. Among them, multilocus sequence typing (MLST) profiling showed the presence of 32 different sequence types (ST). Globally, most of the STs to hospital-adapted high-risk clones and belonged to clonal complexes (CC) associated to the hospital environment, such as CC2. The virulence gene most commonly detected among the strains was altE. High resistance rates to macrolides and aminoglycosides were detected and 64% of the strains harboured the mecA gene, which was codified in SCCmec types. Our results indicates the existence of a complex and genetically diverse S. epidermidis population in the NICU environment. A better knowledge of S. epidermidis strains may help to devise strategies to avoid their conversion from symbiont to pathobiont microorganisms in the NICUs.

Microcins are low-molecular-weight, ribosomally produced, highly stable, bacterial-inhibitory molecules involved in competitive, and amensalistic interactions between Enterobacteriaceae in the intestine. These interactions take place in a highly complex chemical landscape, the intestinal eco-active chemosphere, composed of chemical substances that positively or negatively influence bacterial growth, including those originated from nutrient uptake, and those produced by the action of the human or animal host and the intestinal microbiome. The contribution of bacteria results from their effect on the host generated molecules, on food and digested food, and organic substances from microbial origin, including from bacterial degradation. Here, we comprehensively review the main chemical substances present in the human intestinal chemosphere, particularly of those having inhibitory effects on microorganisms. With this background, and focusing on Enterobacteriaceae, the most relevant human pathogens from the intestinal microbiota, the microcin’s history and classification, mechanisms of action, and mechanisms involved in microcin’s immunity (in microcin producers) and resistance (non-producers) are reviewed. Products from the chemosphere likely modulate the ecological effects of microcin activity. Several cross-resistance mechanisms are shared by microcins, colicins, bacteriophages, and some conventional antibiotics, which are expected to produce cross-effects. Double-microcin-producing strains (such as microcins MccM and MccH47) have been successfully used for decades in the control of pathogenic gut organisms. Microcins are associated with successful gut colonization, facilitating translocation and invasion, leading to bacteremia, and urinary tract infections. In fact, Escherichia coli strains from the more invasive phylogroups (e.g., B2) are frequently microcinogenic. A publicly accessible APD3 database http://aps.unmc.edu/AP/ shows particular genes encoding microcins in 34.1% of E. coli strains (mostly MccV, MccM, MccH47, and MccI47), and much less in Shigella and Salmonella (

In recent years, the etiology of caries has evolved from a simplistic infectious perspective based on Streptococcus mutans and/or Lactobacillus activity, to a multifactorial disease involving a complex oral microbiota, the human genetic background and the environment. The aim of this work was to identify bacterial markers associated with early caries using massive 16S rDNA. To minimize the other factors, the composition of the oral microbiota of twins in which only one of them had caries was compared with their healthy sibling. Twenty-one monozygotic twin pairs without a previous diagnosis of caries were recruited in the context of their orthodontic treatment and divided into two categories: (1) caries group in which only one of the twins had caries; and (2) control group in which neither of the twins had caries. Each participant contributed a single oral lavage sample in which the bacterial composition was determined by 16S rDNA amplification and further high-throughput sequencing. Data analysis included statistical comparison of alpha and beta diversity, as well as differential taxa abundance between groups. Our results show that twins of the control group have a closer bacterial composition than those from the caries group. However, statistical differences were not detected and we were unable to find any particular bacterial marker by 16S rDNA high-throughput sequencing that could be useful for prevention strategies. Although these results should be validated in a larger population, including children from other places or ethnicities, we conclude that the occurrence of caries is not related to the increase of any particular bacterial population.

Adequate gut microbiota establishment is important for lifelong health. The aim was to sequentially analyze the gut microbiota establishment in low-birth-weight preterm neonates admitted to a single neonatal intensive care unit during their first 3 weeks of life, comparing two epidemiological scenarios. Seven control infants were recruited, and another 12 during a severe S. marcescens outbreak. Meconium and feces from days 7, 14, and 21 of life were collected. Gut microbiota composition was determined by 16S rDNA massive sequencing. Cultivable isolates were genotyped by pulsed-field gel electrophoresis, with four S. marcescens submitted for whole-genome sequencing. The expected bacterial ecosystem expansion after birth is delayed, possibly related to antibiotic exposure. The Proteobacteria phylum dominates, although with marked interindividual variability. The outbreak group considerably differed from the control group, with higher densities of Escherichia coli and Serratia to the detriment of Enterococcus and other Firmicutes. Curiously, obligate predators were only detected in meconium and at very low concentrations. Genotyping of cultivable bacteria demonstrated the high bacterial horizontal transmission rate that was confirmed with whole-genome sequencing for S. marcescens. Preterm infants admitted at NICU are initially colonized by homogeneous microbial communities, most of them from the nosocomial environment, which subsequently evolve according to the individual conditions. Our results demonstrate the hospital epidemiology pressure, particularly during outbreak situations, on the gut microbiota establishing process.

Previous studies in experimental autoimmune encephalomyelitis (EAE) models have shown that some probiotic bacteria beneficially impact the development of this experimental disease. Here, we tested the therapeutic effect of two commercial multispecies probiotics—Lactibiane iki and Vivomixx—on the clinical outcome of established EAE. Lactibiane iki improves EAE clinical outcome in a dose-dependent manner and decreases central nervous system (CNS) demyelination and inflammation. This clinical improvement is related to the inhibition of pro-inflammatory and the stimulation of immunoregulatory mechanisms in the periphery. Moreover, both probiotics modulate the number and phenotype of dendritic cells (DCs). Specifically, Lactibiane iki promotes an immature, tolerogenic phenotype of DCs that can directly induce immune tolerance in the periphery, while Vivomixx decreases the percentage of DCs expressing co-stimulatory molecules. Finally, gut microbiome analysis reveals an altered microbiome composition related to clinical condition and disease progression. This is the first preclinical assay that demonstrates that a commercial probiotic performs a beneficial and dose-dependent effect in EAE mice and one of the few that demonstrates a therapeutic effect once the experimental disease is established. Because this probiotic is already available for clinical trials, further studies are being planned to explore its therapeutic potential in multiple sclerosis patients.

ABSTRACT Cystic fibrosis (CF) lung microbiota composition has recently been redefined by the application of next-generation sequencing (NGS) tools, identifying, among others, previously undescribed anaerobic and uncultivable bacteria. In the present study, we monitored the fluctuations of this ecosystem in 15 CF patients during a 1-year follow-up period, describing for the first time, as far as we know, the presence of predator bacteria in the CF lung microbiome. In addition, a new computational model was developed to ascertain the hypothetical ecological repercussions of a prey-predator interaction in CF lung microbial communities. Fifteen adult CF patients, stratified according to their pulmonary function into mild (n = 5), moderate (n = 9), and severe (n = 1) disease, were recruited at the CF unit of the Ramón y Cajal University Hospital (Madrid, Spain). Each patient contributed three or four induced sputum samples during a 1-year follow-up period. Lung microbiota composition was determined by both cultivation and NGS techniques and was compared with the patients’ clinical variables. Results revealed a particular microbiota composition for each patient that was maintained during the study period, although some fluctuations were detected without any clinical correlation. For the first time, Bdellovibrio and Vampirovibrio predator bacteria were shown in CF lung microbiota and reduced-genome bacterial parasites of the phylum Parcubacteria were also consistently detected. The newly designed computational model allows us to hypothesize that inoculation of predators into the pulmonary microbiome might contribute to the control of chronic colonization by CF pathogens in early colonization stages. IMPORTANCE The application of NGS to sequential samples of CF patients demonstrated the complexity of the organisms present in the lung (156 species) and the constancy of basic individual colonization patterns, although some differences between samples from the same patient were observed, probably related to sampling bias. Bdellovibrio and Vampirovibrio predator bacteria were found for the first time by NGS as part of the CF lung microbiota, although their ecological significance needs to be clarified. The newly designed computational model allows us to hypothesize that inoculation of predators into the lung microbiome can eradicate CF pathogens in early stages of the process. Our data strongly suggest that lower respiratory microbiome fluctuations are not necessarily related to the patient’s clinical status.

Background: Several studies have suggested that Helicobacter pylori (H. pylori) infection is a risk factor for colorectal cancer (CRC), while others have not confirmed this hypothesis. This work aimed to assess the relation of CRC with H. pylori seropositivity and with seropositivity to 16 H. pylori proteins, in the MultiCase-Control study, MCC-Spain.Methods: MCC-Spain is a multicase-control study carried out in Spain from 2008 to 2013. In total, 2,140 histologically-confirmed incident CRC cases and 4,098 population-based controls were recruited. Controls were frequency-matched by sex, age, and province. Epidemiological data were collected through a questionnaire fulfilled by face-to-face interviews and a self-administered food-frequency questionnaire. Seroreactivities against 16 H. pylori proteins were determined in 1,488 cases and 2,495 controls using H. pylori multiplex serology. H. pylori seropositivity was defined as positivity to ≥4 proteins. Multivariable logistic regression mixed models were used to estimate odds ratios (OR) and 95% confidence intervals (CI).Results:H. pylori seropositivity was not associated with increased CRC risk (OR = 0.91; 95% CI: 0.71–1.16). Among H. pylori seropositive subjects, seropositivity to Cagδ showed a lower CRC risk, and risk decreased with increasing number of proteins seropositive. Seropositivity to the most recognized virulence factors, CagA and VacA, was not associated with a higher CRC risk. No statistically significant heterogeneity was identified among tumor sites, although inverse relations were stronger for left colon cancer. An interaction with age and sex was found: H. pylori seropositivity was associated with a lower CRC risk in men younger than 65 and with a higher risk in older women.Conclusions: Our results suggest that neither H. pylori seropositivity, nor seropositivity to the virulence factor CagA are associated with a higher CRC risk. A possible effect modification by age and sex was identified.

Increase of extraintestinal pathogenic Escherichia coli (ExPEC) showing resistance to beta-lactams is a major public health concern. This study was conducted as a first molecular epidemiological study on ExPEC in Cuba, regarding prevalence of extended-spectrum beta-lactamases (ESBLs) and carbapenemase genes. A total of 306 ExPEC isolates collected in medical institutions in 16 regions in Cuba (2014−2018) were analyzed for their genotypes and presence of genes encoding ESBL, carbapenemase, plasmid-mediated quinolone resistance (PMQR) determinants by PCR and sequencing. The most common phylogenetic group of ExPEC was B2 (49%), followed by D (23%), A (21%), and B1 (7%). Among ESBL genes detected, blaCTX-M was the most common and detected in 61% of ExPEC, with blaCTX-M-15 being dominant and distributed to all the phylogenetic groups. NDM-1 type carbapenemase gene was identified in two isolates of phylogenetic group B1-ST448. Phylogenetic group B2 ExPEC belonged to mostly ST131 (or its single-locus variant) with O25b allele, harboring blaCTX-M-27, and included an isolate of emerging type ST1193. aac (6’)-Ib-cr was the most prevalent PMQR gene (40.5%), being present in 54.5% of CTX-M-positive isolates. These results indicated high prevalence of CTX-M genes and the emergence of NDM-1 gene among recent ExPEC in Cuba, depicting an alarming situation.

Background: Enterococcus is one of the major human pathogens able to acquire multiple antibiotic-resistant markers as well as virulence factors which also colonize remote ecosystems, including wild animals. In this work, we characterized the Enterococcus population colonizing the gut of Chilean Altiplano camelids without foreign human contact. Material and methods: Rectal swabs from 40 llamas and 10 alpacas were seeded in M-Enterococcus agar, and we selected a total of 57 isolates. Species identification was performed by biochemical classical tests, semi-automated WIDER system, mass spectrometry analysis by MALDI-TOF (matrix-assisted laser desorption/ionization with a time-of-flight mass spectrometer), and, finally, nucleotide sequence of internal fragments of the 16S rRNA, rpoB, pheS, and aac(6)-I genes. Genetic diversity was measured by pulsed field gel electrophoresis (PFGE)-SmaI, whereas the antibiotic susceptibility was determined by the WIDER system. Carriage of virulence factors was explored by polymerase chain reaction (PCR). Results: Our results demonstrated that the most prevalent specie was Enterococcus hirae (82%), followed by other non–Enterococcus faecalis and non–Enterococcus faecium species. Some discrepancies were detected among the identification methods used, and the most reliable were the rpoB, pheS, and aac(6)-I nucleotide sequencing. Selected isolates exhibited susceptibility to almost all studied antibiotics, and virulence factors were not detected by PCR. Finally, some predominant clones were characterized by PFGE into a diverse genetic background. Conclusion: Enterococcus species from the Chilean camelids’ gut microbiota were different from those adapted to humans, and they remained free of antibiotic resistance mechanisms as well as virulence factors.

Objectives: Genetic diversity and resistance of Lactobacillus bulgaricus sbsp. delbrueckii collection with 100 isolates from different home-made yogurt in rural Bulgarian areas were determined. Methods: The strain K98 was the most resistant to bile salts and low pH. Survival and effects on short chain fatty acids production were tested in 20 healthy volunteers. High genetic diversity was observed in the L. bulgaricus collection by RAPD, whereas the ability of tolerate high deoxycholic acid concentrations, and different acid pHs was variable. The strain K98 was selected and used to prepare a homemade yogurt which was administered to 20 healthy volunteers (500 ml/day during 15d). A basal faecal sample and another after yogurt intake were recovered. Results: DGGE experiments, using both universal and Lactic Acid Bacteria (LAB) primers, demonstrated no significant changes in the qualitative composition of gut microbiota. A band corresponding to L. bulgaricus was observed in all 20 samples. Viable L. bulgaricus K98 strain was only recovered in one volunteer. After yogurt intake we found an increase of LAB and Clostridium perfringens, and a decrease of Bacteroides-Prevotella-Porphyromonas. In addition, increases of acetic, butyric and 2-hydroxybutyric acids in faeces were detected. Conclusions: Genetic diversity of L. delbrueckii subsp. bulgaricus especie is high We have isolated a probiotic resistant strain to bile and high acidity, L. delbrueckii subsp. bulgaricus-K98. Qualitative and quantitative changes in the intestinal microbiota are found after ingestion of a homemade yogurt containing this strain, with a concomitant increase in faecal SCFA. Our findings support the interest in developing further studies providing different amounts of L. delbrueckii subsp. bulgaricus-K98, and should evaluate its clinical effects in human disease.

Enterococcus faecium and Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus) were classically clustered into the Lancefield Group D streptococci and despite their taxonomic reclassification still share a similar genetic content and environment. Both species are considered as opportunistic pathogens. E. faecium is often associated with nosocomial bacteraemia, and S. gallolyticus is sporadically found in endocarditis of colorectal cancer patients. In both cases, the source of infection is commonly endogenous with a translocation process that launches through the intestinal barrier. To get new insights into the pathological processes preceding infection development of both organisms, we used an in vitro model with Caco-2 cells to study and compare the adhesion, invasion and translocation inherent abilities of 6 E. faecium and 4 S. gallolyticus well-characterized isolates. Additionally, biofilm formation on polystyrene, collagen I and IV was also explored. Overall results showed that E. faecium translocated more efficiently than S. gallolyticus, inducing a destabilization of the intestinal monolayer. Isolates Efm106, Efm121 and Efm113 (p < .001 compared to Ef222) exhibited the higher translocation ability and were able to adhere 2-3 times higher than S. gallolyticus isolates. Both species preferred the collagen IV coated surfaces to form biofilm but the S. gallolyticus structures were more compact (p = .01). These results may support a relationship between biofilm formation and vegetation establishment in S. gallolyticus endocarditis, whereas the high translocation ability of E. faecium high-risk clones might partially explain the increasing number of bacteraemia.

ABSTRACT Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations. IMPORTANCE With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA.

The characterization of population structures plays a main role for understanding outbreaks and the dynamics of bacterial spreading. In Escherichia coli, the widely used combination of multiplex-PCR scheme together with goeBURST has some limitations. The purpose of this study is to show that the combination of different phylogenetic approaches based on concatenated sequences of MLST genes results in a more precise assignment of E. coli phylogenetic groups, complete understanding of population structure and reconstruction of ancestral clones. A collection of 80 Escherichia coli strains of different origins was analyzed following the Clermont and Doumith's multiplex-PCR schemes. Doumith's multiplex-PCR showed only 1.7% of misassignment, whereas Clermont's-2000 protocol reached 14.0%, although the discrepancies reached 30% and 38.7% respectively when recombinant C, F and E phylogroups were considered. Therefore, correct phylogroup attribution is highly variable and depends on the clonal composition of the sample. As far as population structure of these E. coli strains, including 48 E. coli genomes from GenBank, goeBURST provides a quite dispersed population structure; whereas NeighborNet approach reveals a complex population structure. MLST-based eBURST can infer different founder genotypes, for instance ST23/ST88 could be detected as the founder genotypes for STC23; however, phylogenetic reconstructions might suggest ST410 as the ancestor clone and several evolutionary trajectories with different founders. To improve our routine understanding of E. coli molecular epidemiology, we propose a strategy based on three successive steps; first, to discriminate three main groups A/B1/C, D/F/E and B2 following Doumith's protocol; second, visualization of population structure based on MLST genes according to goeBURST, using NeighborNet to establish more complex relationships among STs; and third, to perform, a cost-free characterization of evolutionary trajectories in variants emerging along the clonal expansion using parsimony methods of phylogenetic analysis.

Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene codes of a chloride channel. The lungs of CF patients are chronically infected by several pathogens but bacteraemia have rarely been reported in this pathology. Besides that, circulating monocytes in CF patients exhibit a patent Endotoxin Tolerance (ET) state since they show a significant reduction of the inflammatory response to bacterial stimulus. Despite a previous description of this phenomenon, the direct cause of ET in CF patients remains unknown. In this study we have researched the possible role of microbial/endotoxin translocation from a localized infection to the bloodstream as a potential cause of ET induction in CF patients. Plasma analysis of fourteen CF patients revealed high levels of LPS compared to healthy volunteers and patients who suffer from Chronic Obstructive Pulmonary Disease. Experiments in vitro showed that endotoxin concentrations found in plasma of CF patients were enough to induce an ET phenotype in monocytes from healthy controls. In agreement with clinical data, we failed to detect bacterial DNA in CF plasma. Our results suggest that soluble endotoxin present in bloodstream of CF patients causes endotoxin tolerance in their circulating monocytes.