The Impact of Colistin Resistance on the Activation of Innate Immunity by Lipopolysaccharide Modification.

Colistin resistance is acquired by different lipopolysaccharide (LPS) modifications. We proposed to evaluate the of effect in vivo colistin resistance acquisition on the innate immune response. We used a pair of ST11 clone Klebsiella pneumoniae strains: an OXA-48, CTX-M-15 K. pneumoniae strain susceptible to colistin (CS-Kp) isolated from a urinary infection and its colistin-resistant variant (CR-Kp) from the same patient after prolonged treatment with colistin. No mutation of previously described genes for colistin resistance (pmrA, pmrB, mgrB, phoP/Q, arnA, arnC, arnT, ugdH, and crrAB) was found in the CR-Kp genome; however, LPS modifications were characterized by negative-ion matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The strains were cocultured with human monocytes to determine their survival after phagocytosis and induction to apoptosis. Also, monocytes were stimulated with bacterial LPS to study cytokine and immune checkpoint production. The addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A of CR-Kp accounted for the colistin resistance. CR-Kp survived significantly longer inside human monocytes after being phagocytosed than did the CS-Kp strain. In addition, LPS from CR-Kp induced both higher apoptosis in monocytes and higher levels of cytokine and immune checkpoint production than LPS from CS-Kp. Our data reveal a variable impact of colistin resistance on the innate immune system, depending on the responsible mechanism. Adding Ara4N to LPS in K. pneumoniae increases bacterial survival after phagocytosis and elicits a higher inflammatory response than its colistin-susceptible counterpart.

Automatic Discrimination of Species within the Enterobacter cloacae Complex Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Supervised Algorithms.

The Enterobacter cloacae complex (ECC) encompasses heterogeneous clusters of species that have been associated with nosocomial outbreaks. These species may have different acquired antimicrobial resistance and virulence mechanisms, and their identification is challenging. This study aims to develop predictive models based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles and machine learning for species-level identification. A total of 219 ECC and 118 Klebsiella aerogenes clinical isolates from three hospitals were included. The capability of the proposed method to differentiate the most common ECC species (Enterobacter asburiae, Enterobacter kobei, Enterobacter hormaechei, Enterobacter roggenkampii, Enterobacter ludwigii, and Enterobacter bugandensis) and K. aerogenes was demonstrated by applying unsupervised hierarchical clustering with principal-component analysis (PCA) preprocessing. We observed a distinctive clustering of E. hormaechei and K. aerogenes and a clear trend for the rest of the ECC species to be differentiated over the development data set. Thus, we developed supervised, nonlinear predictive models (support vector machine with radial basis function and random forest). The external validation of these models with protein spectra from two participating hospitals yielded 100% correct species-level assignment for E. asburiae, E. kobei, and E. roggenkampii and between 91.2% and 98.0% for the remaining ECC species; with data analyzed in the three participating centers, the accuracy was close to 100%. Similar results were obtained with the Mass Spectrometric Identification (MSI) database developed recently (https://msi.happy-dev.fr) except in the case of E. hormaechei, which was more accurately identified with the random forest algorithm. In short, MALDI-TOF MS combined with machine learning was demonstrated to be a rapid and accurate method for the differentiation of ECC species.

Folliculitis Decalvans Has a Heterogeneous Microbiological Signature and Impaired Immunological Response.

BACKGROUND: Folliculitis decalvans (FD) is a rare primary neutrophilic scarring alopecia whose etiology has not been completely elucidated yet. OBJECTIVE: The aim of the study was to determine if the follicular microbiota residing in FD-affected hair follicles had a distinct microbiological signature and if an aberrant immune response was present in the pathogenesis of FD. METHODS: We conducted a cross-sectional study of 10 patients affected by FD. Trichoscopy-guided follicular biopsies were taken from affected and healthy scalp to identify the follicular microbiome using next-generation sequencing. We searched for microbiological biomarkers of FD-affected follicles using the linear discriminant analysis (LDA) effect size (LEfSe) tool. Additionally, peripheral blood mononuclear cells were obtained, and their cytokine production was quantified after incubation with pathogen-associated molecular patterns isolated from patients' biopsies and compared with healthy controls. RESULTS: ß-diversity analysis showed statistically significant differences regarding bacteria comparing follicular microbiota of healthy and FD-affected hairs. Ruminococcaceae, Agathobacter sp., Tyzzerella sp., and Bacteriodales vadin HA21 family were good predictors of disease status. IL-10, TNF-α, and IL-6 levels were significantly decreased in patients after incubation with various strains of bacteria compared with controls. CONCLUSION: FD hair follicles have a specific heterogenous follicular bacterial microbiota signature. Additionally, these patients seem to have an impaired immunological response.

Thiosulfinate-Enriched Allium sativum Extract Exhibits Differential Effects between Healthy and Sepsis Patients: The Implication of HIF-1α.

Garlic (Allium sativum) has historically been associated with antioxidant, immunomodulatory, and microbiocidal properties, mainly due to its richness in thiosulfates and sulfur-containing phytoconstituents. Sepsis patients could benefit from these properties because it involves both inflammatory and refractory processes. We evaluated the effects of thiosulfinate-enriched Allium sativum extract (TASE) on the immune response to bacterial lipopolysaccharide (LPS) by monocytes from healthy volunteers (HVs) and patients with sepsis. We also explored the TASE effects in HIF-1α, described as the key transcription factor leading to endotoxin tolerance in sepsis monocytes through IRAK-M expression. Our results showed TASE reduced the LPS-triggered reactive oxygen species (ROS) production in monocytes from both patients with sepsis and HVs. Moreover, this extract significantly reduced tumor necrosis factor (TNF)-α, interleukin-1ß, and interleukin-6 production in LPS-stimulated monocytes from HVs. However, TASE enhanced the inflammatory response in monocytes from patients with sepsis along with increased expression of human leukocyte antigen-DR. Curiously, these dual effects of TASE on immune response were also found when the HV cohort was divided into low- and high-LPS responders. Although TASE enhanced TNFα production in the LPS-low responders, it decreased the inflammatory response in the LPS-high responders. Furthermore, TASE decreased the HIF-1α pathway-associated genes IRAK-M, VEGFA and PD-L1 in sepsis cells, suggesting HIF-1α inhibition by TASE leads to higher cytokine production in these cells as a consequence of IRAK-M downregulation. The suppression of this pathway by TASE was confirmed in vitro with the prolyl hydroxylase inhibitor dimethyloxalylglycine. Our data revealed TASE's dual effect on monocyte response according to status/phenotype and suggested the HIF-1α suppression as the possible underlying mechanism.

[Impact of an active surveillance program and infection control measures on the incidence of carbapenem-resistant gram-negative bacilli in an intensive care unit]. / Impacto de un programa de vigilancia activa y medidas de control de infecciones sobre la incidencia de bacilos gram negativos resistentes a carbapenems en una unidad de cuidados intensivos.

Hospital-acquired infections caused by carbapenem-resistant Gram-negative bacteria (CRGNB) have been increasingly reported worldwide and are associated with high rates of mortality especially in intensive care units(ICUs). Early identification through rectal surveillance cultures and implementation of infection control measures(ICM) including contact precautions, staff education on cleaning and hand hygiene may reduce the spread of these microorganisms. The aim of this work was to assess the impact of enhanced ICM on CRGNB colonization and to describe the molecular epidemiology of these bacteria in a polyvalent ICU in a tertiary level hospital. A prospective study including audits and active surveillance culture program, with molecular characterization, was conducted before and after the implementation of prevention programs and infection control measures. Microbiological screening was performed in chromogenic media; PCR targeting ß-lactamases genes (blaKPC, blaNDM, blaVIM and blaOXA-48, blaSHV and blaCTX-M), molecular typing by PFGE; and MLST in K. pneumoniae were performed. CRGNB colonization was reduced from 16.92% to 9.67% upon implementing the infection control measures. In K. pneumoniae the most frequent carbapenemase type was KPC-2 associated with SHV-2 and CTX-M-15, and was disseminated in various STs (ST17, ST13, ST2256, ST353); there was no persistence of particular clones and virulence factors showed no association with hypervirulence. IMP-1 carbapenemase predominated in A. baumannii and the PFGE analysis individualized 3 clusters, assuming that the dissemination in the ICU was clonal. The early detection of patients colonized with CRBGN by using epidemiological surveillance cultures and the implementation of prophylactic measures are key to reducing the incidence of these microorganisms.

Characterization of carbapenemase-producing Serratia marcescens and whole-genome sequencing for plasmid typing in a hospital in Madrid, Spain (2016-18).

OBJECTIVES: Carbapenemase-producing Enterobacterales (CPE) are increasingly recognized in nosocomial infections, also affecting ICU patients. We aimed to characterize the carbapenemase-producing Serratia marcescens (CPSm) isolates recovered in our hospital in Madrid (Spain) between March 2016 and December 2018. METHODS: Overall, 50 isolates from clinical and epidemiological surveillance samples were recovered from 24 patients admitted to the medical ICU and 10 non-ICU-related patients based on their phenotypic resistance. Carbapenemase characterization, antibiotic susceptibility, PFGE clonal relatedness, plasmid characterization, WGS (Illumina-NovaSeq 6000) and phylogenetic analysis were performed. RESULTS: A single isolate was finally considered for each patient, except for Patient 8 that was colonized by two different isolates (n = 35). Isolates were characterized as VIM-1 (n = 29) or OXA-48 producers (n = 6). Up to seven genetic lineages were found by PFGE, with dominance of two clones. Plasmid characterization confirmed that almost all CPSm carried the same ∼60 kb IncL OXA-48- or VIM-1-encoding plasmid, which was related to the globally disseminated IncL-pOXA-48a. WGS allowed plasmid reconstruction with two variants: IncL-pVIM-1 (∼65 kb) and IncL-pOXA-48 (∼62 kb). blaOXA-48-Tn1999 (∼5 kb) was the unique antibiotic resistance gene in pOXA-48, whereas pVIM-1 plasmids (∼8 kb) harboured a class 1 integron containing 5'-blaVIM-1+aacA4+dfrB1+aadA1+catB2+qacEDelta1+sul1-3'. CONCLUSIONS: Our results confirm the dissemination of CPSm within our institution in both ICU and non-ICU environments, representing two prevalent CPSm clones, and the same IncL-pOXA-48 plasmid previously described in other Enterobacterales, but containing the blaVIM-1 gene. This also reinforces the relevance of species different from Klebsiella pneumoniae or Escherichia coli in the CPE landscape and circulating lineages and plasmids in local CPE epidemiology.

Murepavadin antimicrobial activity against and resistance development in cystic fibrosis Pseudomonas aeruginosa isolates.

BACKGROUND: Murepavadin, a novel peptidomimetic antibiotic, is being developed as an inhalation therapy for treatment of Pseudomonas aeruginosa respiratory infection in people with cystic fibrosis (CF). It blocks the activity of the LptD protein in P. aeruginosa causing outer membrane alterations. OBJECTIVES: To determine the in vitro activity of murepavadin against CF P. aeruginosa isolates and to investigate potential mechanisms of resistance. METHODS: MIC values were determined by both broth microdilution and agar dilution and results compared. The effect of artificial sputum and lung surfactant on in vitro activity was also measured. Spontaneous mutation frequency was estimated. Bactericidal activity was investigated using time-kill assays. Resistant mutants were studied by WGS. RESULTS: The murepavadin MIC50 was 0.125 versus 4 mg/L and the MIC90 was 2 versus 32 mg/L by broth microdilution and agar dilution, respectively. Essential agreement was >90% when determining in vitro activity with artificial sputum or lung surfactant. It was bactericidal at a concentration of 32 mg/L against 95.4% of the strains within 1-5 h. Murepavadin MICs were 2-9 two-fold dilutions higher for the mutant derivatives (0.5 to >16 mg/L) than for the parental strains. Second-step mutants were obtained for the PAO mutS reference strain with an 8×MIC increase. WGS showed mutations in genes involved in LPS biosynthesis (lpxL1, lpxL2, bamA2, lptD, lpxT and msbA). CONCLUSIONS: Murepavadin characteristics, such as its specific activity against P. aeruginosa, its unique mechanism of action and its strong antimicrobial activity, encourage the further clinical evaluation of this drug.

Anti-biofilm activity of murepavadin against cystic fibrosis Pseudomonas aeruginosa isolates.

OBJECTIVES: To determine the activity of murepavadin in comparison with tobramycin, colistin and aztreonam, against cystic fibrosis (CF) Pseudomonas aeruginosa isolates growing in biofilms. The biofilm-epidemiological cut-off (ECOFF) values that include intrinsic resistance mechanisms present in biofilms were estimated. METHODS: Fifty-three CF P. aeruginosa isolates from respiratory samples were tested using the Calgary (closed system) device, while 4 [2 clinical (one smooth, one mucoid) and 2 reference strains] were tested using the BioFlux, a microfluidic open model of biofilm testing. Biofilm was stained with SYTO9® and propidium iodide. The minimal biofilm inhibitory concentration (MBIC) and the minimal biofilm eradication concentration (MBEC) were determined. The MBIC-ECOFF and the MBEC-ECOFF were calculated. RESULTS: Colistin, tobramycin and murepavadin presented similar MBIC50/MBIC90 values (4/32, 8/64 and 2/32, respectively). Murepavadin exhibited the lowest MBEC90 (64 mg/L). Aztreonam MBIC and MBEC values were higher than those of the other antibiotics tested. Tobramycin and murepavadin had the lowest MBEC-ECOFF (64 and 128 mg/L, respectively), while those of aztreonam and colistin exceeded 512 mg/L. Using the BioFlux, for the PAO1, PAO mutS and the smooth clinical strain, a significant difference (P < 0.0125) was observed when comparing the fluorescence of treated and untreated biofilms. For the mucoid strain, only the biofilm treated with aztreonam (MBIC and MBEC) and tobramycin (MBEC) showed differences with respect to the untreated biofilm. CONCLUSIONS: Murepavadin demonstrated good activity against P. aeruginosa biofilms both in open and closed systems. The MBIC-ECOFF and the MBEC-ECOFF are proposed as new parameters to estimate the activity of antibiotics on biofilms.

Evaluation of CHROMagar™-Serratia agar, a new chromogenic medium for the detection and isolation of Serratia marcescens.

A comparative analysis of the performance of the new selective chromogenic CHROMagar™-Serratia culture medium for detection and isolation of Serratia marcescens was undertaken. A total of 134 clinical isolates (95 S. marcescens with and without carbapenemase production and 39 non-S. marcescens isolates) and 96 epidemiological samples (46 rectal swabs and 50 from environmental surfaces) were studied. Diagnostic values when compared with CHROMagar™-Orientation medium were 96.8% sensitivity, 100% specificity, 100% positive predictive value and 88.5% negative predictive value. In conclusion, CHROMagar™-Serratia shows an excellent ability for differentiation of S. marcescens among clinical isolates and in environmental samples.

Prevalence and risks factors associated with ESBL-producing faecal carriage in a single long-term-care facility in Spain: emergence of CTX-M-24- and CTX-M-27-producing Escherichia coli ST131-H30R.

OBJECTIVES: To address the faecal carriage prevalence of antibiotic-multiresistant bacteria and associated risk factors in a public long-term care facility (LTCF). METHODS: A prospective study in a single government-funded LTCF of 300 residents in Ciudad Real, Spain. Residents' clinical and demographic data were collected, as well as recent antibiotic consumption in the institution. Each participant contributed a rectal swab, which was plated on selective and differential-selective media. Colonies were identified by MALDI-TOF and ESBL production was confirmed by the double-disc synergy method, with characterization of the molecular mechanism by PCR. Isolates were typed by PFGE and submitted for ST131 screening by PCR. RESULTS: Faecal carriage of ESBL-producing Enterobacterales was detected in 58 (31%) of 187 participants and previous infection by MDR bacteria was identified as a risk factor. The genes characterized were: blaCTX-M-15 (40.6%); blaCTX-M-14 (28.8%); blaCTX-M-27 (13.5%); and blaCTX-M-24 (10.1%). Some 56.4% of the isolates were grouped into the E. coli ST131 clone; 70.9% of these corresponded to the O25b serotype, 51.6% of them to Clade C1 (H30) and 12.9% to Clade C2 (H30Rx). Clade C1 isolates were mostly C1-M27, whereas the C2 sublineage was mainly related to the production of CTX-M-15. ST131-CTX-M-24 isolates (n = 6) corresponded to Clade A with serotype O16. CONCLUSIONS: A high prevalence of ESBL-producing Enterobacterales faecal carriage has been detected in a single LTCF, highlighting the emergence of ST131 Clade A-M24 and Clade C1-M27 lineages.

Intestinal Immune Dysregulation Driven by Dysbiosis Promotes Barrier Disruption and Bacterial Translocation in Rats With Cirrhosis.

In cirrhosis, intestinal dysbiosis, intestinal barrier impairment, and systemic immune system abnormalities lead to gut bacterial translocation (GBT) and bacterial infection. However, intestinal immune system dysfunction and its contribution to barrier damage are poorly understood. This study correlates immune system dysregulation in the intestines of rats at different stages of CCl4 -induced cirrhosis with barrier function and pathogenic microbiota. The following variables were addressed in the small intestine: intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) activation status and cytokine production (flow cytometry), cytokine mRNA and protein expression (quantitative real-time PCR and immunofluorescence), microbiota composition of ileum content (16S recombinant DNA massive sequencing), permeability (fecal albumin loss), and epithelial junctions (immunohistochemistry and immunofluorescence). The intestinal mucosa in rats with cirrhosis showed a proinflammatory pattern of immune dysregulation in IELs and LPLs, which featured the expansion of activated lymphocytes, switch to a T helper 1 (Th1) regulatory pattern, and Th17 reduction. In rats with cirrhosis with ascites, this state was associated with epithelial junction protein disruption, fecal albumin loss, and GBT. Direct correlations (P < 0.01) were observed between elevated interferon gamma (IFNγ)-expressing T cytotoxic LPLs and fecal albumin and between inflammatory taxa abundance and IFNγ-producing immune cells in the ileum. Bowel decontamination led to redistributed microbiota composition, reduced proinflammatory activation of mucosal immune cells, normalized fecal albumin levels, and diminished GBT; but there were no modifications in Th17 depletion. Conclusion: The intestinal mucosa of rats with cirrhosis acquires a proinflammatory profile of immune dysregulation that parallels the severity of cirrhosis; this impaired intestinal immune response is driven by gut dysbiosis and leads to disrupted barrier function, promoting GBT.

ZEB1 promotes inflammation and progression towards inflammation-driven carcinoma through repression of the DNA repair glycosylase MPG in epithelial cells.

OBJECTIVE: Chronic inflammation is a risk factor in colorectal cancer (CRC) and reactive oxygen species (ROS) released by the inflamed stroma elicit DNA damage in epithelial cells. We sought to identify new drivers of ulcerative colitis (UC) and inflammatory CRC. DESIGN: The study uses samples from patients with UC, mouse models of colitis and CRC and mice deficient for the epithelial-to-mesenchymal transition factor ZEB1 and the DNA repair glycosylase N-methyl-purine glycosylase (MPG). Samples were analysed by immunostaining, qRT-PCR, chromatin immunoprecipitation assays, microbiota next-generation sequencing and ROS determination. RESULTS: ZEB1 was induced in the colonic epithelium of UC and of mouse models of colitis. Compared with wild-type counterparts, Zeb1-deficient mice were partially protected from experimental colitis and, in a model of inflammatory CRC, they developed fewer tumours and exhibited lower levels of DNA damage (8-oxo-dG) and higher expression of MPG. Knockdown of ZEB1 in CRC cells inhibited 8-oxo-dG induction by oxidative stress (H2O2) and inflammatory cytokines (interleukin (IL)1ß). ZEB1 bound directly to the MPG promoter whose expression inhibited. This molecular mechanism was validated at the genetic level and the crossing of Zeb1-deficient and Mpg-deficient mice reverted the reduced inflammation and tumourigenesis in the former. ZEB1 expression in CRC cells induced ROS and IL1ß production by macrophages that, in turn, lowered MPG in CRC cells thus amplifying a positive loop between both cells to promote DNA damage and inhibit DNA repair. CONCLUSIONS: ZEB1 promotes colitis and inflammatory CRC through the inhibition of MPG in epithelial cells, thus offering new therapeutic strategies to modulate inflammation and inflammatory cancer.

Phylogenomics of Enterococcus faecalis from wild birds: new insights into host-associated differences in core and accessory genomes of the species.

Wild birds have been suggested to be reservoirs of antimicrobial resistant and/or pathogenic Enterococcus faecalis (Efs) strains, but the scarcity of studies and available sequences limit our understanding of the population structure of the species in these hosts. Here, we analysed the clonal and plasmid diversity of 97 Efs isolates from wild migratory birds. We found a high diversity, with most sequence types (STs) being firstly described here, while others were found in other hosts including some predominant in poultry. We found that pheromone-responsive plasmids predominate in wild bird Efs while 35% of the isolates entirely lack plasmids. Then, to better understand the ecology of the species, the whole genome of fivestrains with known STs (ST82, ST170, ST16 and ST55) were sequenced and compared with all the Efs genomes available in public databases. Using several methods to analyse core and accessory genomes (AccNET, PLACNET, hierBAPS and PANINI), we detected differences in the accessory genome of some lineages (e.g. ST82) demonstrating specific associations with birds. Conversely, the genomes of other Efs lineages exhibited divergence in core and accessory genomes, reflecting different adaptive trajectories in various hosts. This pangenome divergence, horizontal gene transfer events and occasional epidemic peaks could explain the population structure of the species.

[Characterization of Salmonella Heidelberg strains isolated in Chile]. / Caracterización de cepas clínicas y ambientales de Salmonella enterica subsp. enterica serovar Heidelberg aisladas en Chile.

BACKGROUND: Salmonella Heidelberg (S. Heidelberg) causes gastroenteritis and sometimes bacteremia and endocarditis. In other countries, this serovar has multidrug resistance including extended-spectrum ß-lactamases (ESBLs) and AmpC (ß-lactamases (AmpC), associated with the blaCMY-2 gene. In Chile, an outbreak by S. Heidelberg occurred in 2011, the phenotypic and genetic characteristics of Chilean strains are unknown. AIM: To determine the antimicrobial susceptibility, presence of plasmids and virulence factor genes in S. Heidelberg strains isolated in Chile over the period 2006-2011. MATERIAL AND METHODS: In sixty-one S. Heidelberg clinical and environmental strains collected by the Public Health Institute in Chile during 2006-2011, antimicrobial susceptibility, plasmids and virulence factor genes (invA, sifA, pefA, agfA, lpfA and, stkD) were studied. RESULTS: S. Heidelberg had a high susceptibility to sulfamethoxazole-trimethoprim, gentamicin, ceftriaxone, ceftiofur, chloramphenicol, amoxicillin-clavulanic acid and ampicillin. However, 52% had decreased susceptibility to ciprofloxacin and 33% resistance to tetracycline. ESBLs were detected in three strains isolated from blood cultures, environment and human feces. The latter strain was positive for AmpC and blaCMY-2 gene. Fifty three of 61 strains showed one to seven plasmids of 0.8 to approximately 30 kb. Most plasmids were small with sizes between 0.8 and 2 kb. All isolates were positive for all genes except pefA. CONCLUSIONS: S. Heidelberg isolated from Chilean samples was susceptible to first-line antimicrobials, except tetracycline and ciprofloxacin. The emergence of strains with ESBLs and AmpC should be a warning. The strains were homogeneous for virulence genes, but heterogeneous in their plasmids.

Interplay among Resistance Profiles, High-Risk Clones, and Virulence in the Caenorhabditis elegans Pseudomonas aeruginosa Infection Model.

The increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa is frequently linked to widespread international strains designated high-risk clones. In this work, we attempted to decipher the interplay between resistance profiles, high-risk clones, and virulence, testing a large (n = 140) collection of well-characterized P. aeruginosa isolates from different sources (bloodstream infections, nosocomial outbreaks, cystic fibrosis, and the environment) in a Caenorhabditis elegans infection model. Consistent with previous data, we documented a clear inverse correlation between antimicrobial resistance and virulence in the C. elegans model. Indeed, the lowest virulence was linked to XDR profiles, which were typically linked to defined high-risk clones. However, virulence varied broadly depending on the involved high-risk clone; it was high for sequence type 111 (ST111) and ST235 but very low for ST175. The highest virulence of ST235 could be attributed to its exoU+ type III secretion system (TTSS) genotype, which was found to be linked with higher virulence in our C. elegans model. Other markers, such as motility or pigment production, were not essential for virulence in the C. elegans model but seemed to be related with the higher values of the statistical normalized data. In contrast to ST235, the ST175 high-risk clone, which is widespread in Spain and France, seems to be associated with a particularly low virulence in the C. elegans model. Moreover, the previously described G154R AmpR mutation, prevalent in ST175, was found to contribute to the reduced virulence, although it was not the only factor involved. Altogether, our results provide a major step forward for understanding the interplay between P. aeruginosa resistance profiles, high-risk clones, and virulence.

Antibody reactivity against Helicobacter pylori proteins in a sample of the Spanish adult population in 2008-2013.

BACKGROUND: Differences in Helicobacter pylori protein expression have been related to the risk of severe gastric diseases. In Spain, a marked geographic pattern in gastric cancer mortality has long been reported. OBJECTIVE: To characterize antibody reactivity patterns against 16 H. pylori proteins, by age, sex, and region of birth, in a large sample of the Spanish adult population. MATERIALS AND METHODS: Antibody reactivity was quantified by H. pylori multiplex serology in a sample from the control group of the multicase-control study MCC-Spain. For this analysis, 2555 population-based controls were included. Each participant was classified as seropositive or seronegative for each protein according to specific cutoffs. Overall H. pylori seroprevalence was defined as positivity against ≥4 proteins. Descriptive analyses by age, sex, and region of birth were performed for both seroprevalence and seroreactivity (continuous measure). Differences among groups were tested by logistic and linear regression models. RESULTS: Overall H. pylori seroprevalence increased with age in both sexes. For ages 55-74, seroprevalence was lower in women than in men (84% vs 92%, P<.001). Region of birth explained 7% of the variability in seroprevalence. Among H. pylori seropositive subjects, proteins with the highest seroprevalence were GroEL, NapA, HP231, and Omp. Seropositivity for most of the proteins increased or remained stable with age, rising mainly for CagA, GroEL, and HyuA in women. A clear cohort effect was not observed. CONCLUSIONS: This is the first study to describe the antibody patterns against 16 H. pylori proteins in the Spanish population. We found variability in the H. pylori antibody profiles according to both individual factors such as age and sex, and environmental factors such as the region of birth. The slightness of the reduction in seropositivity with decreasing age highlights the ongoing importance of this infection.

Association of Streptococcus gallolyticus subspecies gallolyticus with colorectal cancer: Serological evidence.

The colonic opportunist Streptococcus gallolyticus subspecies gallolyticus (SGG) is potentially associated with colorectal cancer (CRC). Large-scale seroepidemiological data for SGG antibodies and their possible association with CRC is currently missing. Associations between CRC and antibody responses to SGG were examined in 576 CRC cases and 576 controls matched by sex, age and province from a population-based multicase-control project (MCC-Spain). MCC-Spain was conducted between 2008 and 2013 in 12 Spanish provinces. Antibody responses to recombinant affinity-purified SGG pilus proteins Gallo1569, 2039, 2178 and 2179 were analysed by multiplex serology. Polyomavirus (PyV) JC VP1 and PyV 6 VP1 proteins served as disease-specificity controls. In the control population, antibody responses to pilus proteins were mostly weak. Antibody responses to individual pilus proteins Gallo2039 (OR: 1.58, 95% CI: 1.09-2.28), Gallo2178 (OR: 1.58, 95% CI: 1.09-2.30) and Gallo2179 (OR: 1.45, 95% CI: 1.00-2.11) were significantly associated with CRC risk. The association was stronger for positivity to two or more pilus proteins of Gallo1569, Gallo2178 and Gallo2179 (OR:1.93, 95% CI: 1.04-3.56) and for double-positivity to Gallo2178 and Gallo2179 (OR: 3.54, 95% CI: 1.49-8.44). The association between SGG infection and CRC risk was stronger among individuals younger than 65 years. For the first time we demonstrated a statistically significant association of exposure to SGG antigens and CRC in a large seroepidemiological study. These results should stimulate further studies on the role of SGG in CRC pathogenesis.

Obeticholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats.

BACKGROUND & AIMS: In advanced cirrhosis, gut bacterial translocation is the consequence of intestinal barrier disruption and leads to bacterial infection. Bile acid abnormalities in cirrhosis could play a role in the integrity of the intestinal barrier and the control of microbiota, mainly through the farnesoid X receptor. We investigated the long-term effects of the farnesoid X receptor agonist, obeticholic acid, on gut bacterial translocation, intestinal microbiota composition, barrier integrity and inflammation in rats with CCl4-induced cirrhosis with ascites. METHODS: Cirrhotic rats received a 2-week course of obeticholic acid or vehicle starting once ascites developed. We then determined: bacterial translocation by mesenteric lymph node culture, ileum expression of antimicrobial peptides and tight junction proteins by qPCR, fecal albumin loss, enteric bacterial load and microbiota composition by qPCR and pyrosequencing of ileum mucosa-attached contents, and intestinal inflammation by cytometry of the inflammatory infiltrate. RESULTS: Obeticholic acid reduced bacterial translocation from 78.3% to 33.3% (p<0.01) and upregulated the expression of the farnesoid X receptor-associated gene small heterodimer partner. Treatment improved ileum expression of antimicrobial peptides, angiogenin-1 and alpha-5-defensin, tight junction proteins zonulin-1 and occludin, and reduced fecal albumin loss and liver fibrosis. Enteric bacterial load normalized, and the distinctive mucosal microbiota of cirrhosis was reduced. Gut immune cell infiltration was reduced and inflammatory cytokine and Toll-like receptor 4 expression normalized. CONCLUSIONS: In ascitic cirrhotic rats, obeticholic acid reduces gut bacterial translocation via several complementary mechanisms at the intestinal level. This agent could be used as an alternative to antibiotics to prevent bacterial infection in cirrhosis.

Evaluation of bacteriocinogenic activity, safety traits and biotechnological potential of fecal lactic acid bacteria (LAB), isolated from Griffon Vultures (Gyps fulvus subsp. fulvus).

BACKGROUND: Lactic acid bacteria (LAB) are part of the gut microbiota and produce ribosomally synthesized antimicrobial peptides or bacteriocins with interest as natural food preservatives and therapeutic agents. Bacteriocin-producing LAB are also attractive as probiotics. Griffon vultures (Gyps fulvus subspecies fulvus) are scavenger birds that feed almost exclusively on carrion without suffering apparent ill effects. Therefore, griffon vultures might be considered a reservoir of bacteriocin-producing lactic acid bacteria (LAB) with potential biotechnological applications. RESULTS: Griffon vulture feces were screened for LAB with antimicrobial activity, genes encoding bacteriocins, potential virulence determinants, susceptibility to antibiotics, genotyping and characterization of bacteriocins. In this study, from 924 LAB evaluated 332 isolates (36 %) showed direct antimicrobial activity against Gram-positive bacteria only. The molecular identification of the most antagonistic 95 isolates showed that enterococci was the largest LAB group with antimicrobial activity (91 %) and E. faecium (40 %) the most identified antagonistic species. The evaluation of the presence of bacteriocin structural genes in 28 LAB isolates with the highest bacteriocinogenic activity in their supernatants determined that most enterococcal isolates (75 %) encoded multiple bacteriocins, being enterocin A (EntA) the largest identified (46 %) bacteriocin. Most enterococci (88 %) were resistant to multiple antibiotics. ERIC-PCR and MLST techniques permitted genotyping and recognition of the potential safety of the bacteriocinogenic enterococci. A multiple-step chromatographic procedure, determination of the N-terminal amino acid sequence of purified bacteriocins by Edman degradation and a MALDI TOF/TOF tandem MS procedure permitted characterization of bacteriocins present in supernatants of producer cells. CONCLUSIONS: Enterococci was the largest LAB group with bacteriocinogenic activity isolated from griffon vulture feces. Among the isolates, E. faecium M3K31 has been identified as producer of enterocin HF (EntHF), a bacteriocin with remarkable antimicrobial activity against most evaluated Listeria spp. and of elevated interest as a natural food preservative. E. faecium M3K31 would be also considered a safe probiotic strain for use in animal nutrition.