Behaviour of Bdellovibrio bacteriovorus in the presence of Gram-positive Staphylococcus aureus.

The present study aimed to characterize the behavior of Bdellovibrio bacteriovorus in the presence of Staphylococcus aureus. B. bacteriovorus was co-cultured with S. aureus or Pseudomonas aeruginosa or Streptococcus mutans, in planktonic and sessile conditions. Co-cultures were studied by Field-Emission Scanning Electron Microscopy (FESEM), Scanning Transmission Electron Microscopy (STEM), turbidimetry, quantitative PCR (qPCR), and sequencing of gene Bd0108 of B. bacteriovorus. Results indicated that B. bacteriovorus comparably inhibited planktonic growth of P. aeruginosa and S. aureus, but not of S. mutans. FESEM and STEM showed that B. bacteriovorus interacts with S. aureus affecting its cell wall and membrane. Sequencing of gene Bd0108 did not reveal any of the mutations that can arise from the host-interaction (hit) locus. Although some Gram-negative species are reported to be B. bacteriovorus prey, it seems that in case of nutrient deficiency this predatory bacterium can also take advantage of some Gram-positive species. B. bacteriovorus behaviour in the presence of S. aureus is relevant for its possible therapeutic use in several pathologies, like cystic fibrosis in which S. aureus and P. aeruginosa frequently coexist as infectious agents.

Human Babesiosis, Bolivia, 2013.

To investigate human babesiosis in the Bolivian Chaco, in 2013 we tested blood samples from 271 healthy persons living in 2 rural communities in this region. Microscopy and PCR indicated that 3.3% of persons were positive for Babesia microti parasites (US lineage); seroprevalence was 45.7%. Appropriate screening should mitigate the risk for transfusion-associated babesiosis.

Malaria in children of Tshimbulu (Western Kasai, Democratic Republic of the Congo): epidemiological data and accuracy of diagnostic assays applied in a limited resource setting.

BACKGROUND: The literature data on malaria in Western Kasai, DRC, are limited and inadequate. A recent molecular survey there has detected Plasmodium ovale and Plasmodium malariae as mixed infections with Plasmodium falciparum. In Tshimbulu, Western Kasai, during a humanitarian initiative designed to provide children with free preventive screening and to reduce the local high malaria death rate, accurate species identification was performed, in order to collect unambiguous epidemiological data and to evaluate the reliability of locally applied diagnostics. METHODS: Finger pricks provided fresh blood for microscopic analysis (MA), for rapid diagnostic test (RDT) and for molecular diagnostics (MD). MA and RDT were first performed by the local team and then a re-interpretation of the results (on the same slides and on RDT's taken pictures) was conducted in Italy, where MD were performed. RESULTS: The analysis was conducted on 306 children; RDT found 80.9 % as P. falciparum-positive (37.4 % as two-band positive, P. falciparum single infection). MA identified a further four children as positive to P. falciparum and six co-infections with P. ovale. The second RDT evaluation confirmed a similar infection rate (78.2 %) but interpreted as two-band positive a significantly higher share of tests (56.8 %). MA confirmed 80.0 % of the children as malaria positive and, in addition to P. falciparum, identified P. malariae (13.8 %), P. vivax (3.4 %) and P. ovale (2.4 %), and detected Babesia microti in 19 smears. MD confirmed all of the species found (Babesia microti included), classified as mono-infection with P. falciparum a rate of spots comparable to MA revision, and identified all P. ovale as Plasmodium ovale wallikeri. The RDT used locally proved 93.1 % sensitive and 92.1 % specific for P. falciparum. CONCLUSIONS: The malaria prevalence among the children and the presence of four Plasmodium species, highlighted in this study, identified a sanitary issue which proved to be more alarming than expected, as it was worsened by the unpredictable presence of P. vivax and Babesia microti (never before reported in DRC). Each diagnostic tool showed its point of weakness. Therefore, the most correct approach is by the combined use of different, locally available, diagnostic tools.

Eubiosis and dysbiosis: the two sides of the microbiota.

The microbial ecosystem of the gastrointestinal tract is characterized by a great number of microbial species living in balance by adopting mutualistic strategies. The eubiosis/dysbiosis condition of the gut microbiota strongly influences our healthy and disease status. This review briefly describes microbiota composition and functions, to then focus on eubiosis and dysbiosis status: the two sides of the microbiota.

Women Skin Microbiota Modifications during Pregnancy.

Several studies have shown fluctuations in the maternal microbiota at various body sites (gut, oral cavity, and vagina). The skin microbiota plays an important role in our health, but studies on the changes during pregnancy are limited. Quantitative and qualitative variations in the skin microbiota in pregnant woman could indeed play important roles in modifying the immune and inflammatory responses of the host. These alterations could induce inflammatory disorders affecting the individual's dermal properties, and could potentially predict infant skin disorder in the unborn. The present study aimed to characterize skin microbiota modifications during pregnancy. For this purpose, skin samples were collected from 52 pregnant women in the first, second, and third trimester of non-complicated pregnancies and from 17 age- and sex-matched healthy controls. The skin microbiota composition was assessed by next generation sequencing (NGS) of the V3-V4 region of the bacterial rRNA 16S. Our results indicate that from the first to the third trimester of pregnancy, changes occur in the composition of the skin microbiota, microbial interactions, and various metabolic pathways. These changes could play a role in creating more advantageous conditions for fetal growth.

Microevolution in fimH gene of mucosa-associated Escherichia coli strains isolated from pediatric patients with inflammatory bowel disease.

Several studies reported increased numbers of mucosa-associated Escherichia coli strains in patients with inflammatory bowel disease (IBD), encompassing Crohn's disease (CD) and ulcerative colitis (UC). The majority of E. coli strains possess type 1 fimbriae, whose tip fibrillum protein, FimH, naturally undergoes amino acid replacements, an important process in the adaptation of commensal E. coli strains to environmental changes, like those observed in IBD and urinary tract infections. In this study, we analyzed mutational patterns in the fimH gene of 52 mucosa-associated E. coli strains isolated from IBD and non-IBD pediatric patients, in order to investigate microevolution of this genetic trait. FimH-positive strains were also phylogenetically typed and tested for their adhesive ability on Caco-2 cells. Specific FimH alleles for each grouping feature were found. Mutations G66S and V27A were related to CD, while mutations A242V, V163A, and T74I were attributed to UC. Otherwise, the G66S, N70S, and S78N mutations were specifically attributed to B2/D phylogroups. The N70S and A119V mutations were related to adhesive E. coli strains. Phylogroup B2, adhesive, and IBD E. coli strains showed a higher site substitution rate (SSR) in the fimH gene, together with a higher number of mutations. The degree of naïve mucosal inflammation was related to specific FimH alleles. Moreover, we could suggest that the V27A mutation is pathoadaptive for the CD intestinal habitat, while we could also suggest that both the N70S and S78N mutations are related to the more virulent E. coli B2 phylogroup. In conclusion, we found some FimH variants that seem to be more involved than others in the evolution of IBD pathogenesis.

A potential role of Escherichia coli pathobionts in the pathogenesis of pediatric inflammatory bowel disease.

Through genomic analysis of mucosa-associated Escherichia coli strains, we found a close genetic association among isolates from pediatric inflammatory bowel disease (IBD) patients. A specific E. coli pathovar, adherent-invasive E. coli (AIEC), was found in Crohn's disease (CD) adult patients - this pathovar has enhanced adhesive and invasive properties, mainly due to the mannose-bonding FimH protein. We aimed to characterize 52 mucosa-associated E. coli strains isolated from pediatric IBD and non-IBD patients. Eleven E. coli strains, showing a strong similarity in fimH gene sequence to that of E. coli AIEC LF82, were characterized for fimH gene sequence, genomic profiling, adhesive and invasive ability, and phylogrouping. The results were compared with E. coli strains AIEC LF82 and MG1655. The 11 E. coli isolates showed 82.4% ± 1.4% fimH sequence similarity and 80.6% ± 1.3% genomic similarity to strain AIEC LF82. All these strains harbored V27A and S78N FimH mutations, as found in LF82. Nine of them belonged to the more virulent B2 and D phylogroups. Neuraminidase treatment, mimicking inflamed mucosa, enhanced adhesion of all 11 strains by 3.5-fold, but none showed invasion ability. It could be argued that the 11 selected strains could be a branch of an E. coli subpopulation (pathobionts), that could take advantage in an inflamed context because of a suitable genomic and (or) genetic backdrop.

A distinctive 'microbial signature' in celiac pediatric patients.

BACKGROUND: Celiac Disease (CD) is an autoimmune disorder of the small intestine in which dietary gluten ingestion leads to a chronic enteropathy. Recently, scientific evidence suggested a potential role of gut microbiota in CD. To have a snapshot of dominant duodenal microbiota we analyzed the mucosa-associated microbiota of 20 children with CD, before and after a gluten-free diet (GFD) regimen, and of 10 controls. Total DNA was extracted from duodenal biopsies and amplification products of 16S ribosomal DNA were compared by temporal temperature gradient gel electrophoresis (TTGE). TTGE profiles were analyzed by statistical multivariate analysis. RESULTS: The average number of bands in TTGE profiles was significantly higher (P < 0.0001) in active (n.b. 16.7 +/- 0.7) and inactive states (n.b. 13.2 +/- 0.8) than in controls (n.b. 3.7 +/- 1.3). Mean interindividual similarity index was 54.9% +/- 14.9% for active disease, 55.6% +/- 15.7% for remission state and 21.8% +/- 30.16% for controls. Similarity index between celiac children before and after GFD treatment was 63.9% +/- 15.8%. Differences in microbiota biodiversity were among active and remission state (P = 0.000224) and amid active CD and controls (P < 0.001). Bacteroides vulgatus and Escherichia coli were detected more often in CD patients than in controls (P < 0.0001). CONCLUSIONS: Overall, the results highlighted a peculiar microbial TTGE profile and a significant higher biodiversity in CD pediatric patients' duodenal mucosa. The possible pathophysiological role of these microbial differences needs further characterization.

Rebuilding the Gut Microbiota Ecosystem.

A microbial ecosystem in which bacteria no longer live in a mutualistic association is called dysbiotic. Gut microbiota dysbiosis is a condition related with the pathogenesis of intestinal illnesses (irritable bowel syndrome, celiac disease, and inflammatory bowel disease) and extra-intestinal illnesses (obesity, metabolic disorder, cardiovascular syndrome, allergy, and asthma). Dysbiosis status has been related to various important pathologies, and many therapeutic strategies aimed at restoring the balance of the intestinal ecosystem have been implemented. These strategies include the administration of probiotics, prebiotics, and synbiotics; phage therapy; fecal transplantation; bacterial consortium transplantation; and a still poorly investigated approach based on predatory bacteria. This review discusses the various aspects of these strategies to counteract intestinal dysbiosis.

Canine Trypanosoma cruzi infection in the Bolivian Chaco.

A cross-sectional study on Trypanosoma cruzi was carried out in 2013 to evaluate the role of dogs as possible source of infection for humans in two rural communities of the highly endemic Bolivian Chaco (Bartolo, Chuquisaca Department, n = 57 dogs; and Ivamirapinta, Santa Cruz Department, n = 48 dogs). Giemsa-stained thick and thin smears, rapid immunochromatographic test (ICT) (Chagas Quick test, Cypress Diagnostic, Belgium) and polymerase chain reaction for T. cruzi on dried blood spots were performed. All smears proved negative by microscopic examination, whereas 23/103 (22%) were positive by ICT and 5/105 (5%) blood samples contained T. cruzi DNA, evidencing the potential role of dogs in the domestic transmission of the parasite.

Intestinal parasitic infections and associated epidemiological drivers in two rural communities of the Bolivian Chaco.

INTRODUCTION: In 2013 a coproparasitological survey was carried out in two rural communities of the Bolivian Chaco to determine the prevalence of intestinal parasitic infections (IPIs) and to investigate on possible infection drivers through a questionnaire interview. METHODOLOGY: Faecal samples were examined by microscopy. Samples positive for Entamoeba histolytica complex and Blastocystis were molecularly examined to identify the species/subtypes involved. RESULTS: The overall infection rate was 86%, identical in both communities and mostly due to protozoa. Soil-transmitted helminths were detected in <3% of children and adults. DISCUSSION: The protozoa detected, including Blastocystis subtypes, indicate faecal contamination of the environment by both humans (as confirmed by the presence of Hymenolepis nana) and animals. Nested-PCR identified E. histolytica, thus signalling the possible occurrence of invasive amoebosis. Lack of safe water, environmental contamination, poor sanitation and hygiene, shared by both communities, are the main drivers of IPIs. In addition, unlike gender and socioeconomic factors, childhood (only for some species), crowding and cohabitation with animals proved to be further significant protozoon infection risk factors. CONCLUSIONS: These results highlight the need for the promotion of access to clean water, improved sanitation and better hygiene, thus reducing the frequency of preventive chemotherapy for STHs while continuing to monitor the population for possible recrudescence.

Gut microbiota related to Giardia duodenalis, Entamoeba spp. and Blastocystis hominis infections in humans from Côte d'Ivoire.

INTRODUCTION: Literature data provide little information about protozoa infections and gut microbiota compositional shifts in humans. This preliminary study aimed to describe the fecal bacterial community composition of people from Côte d'Ivoire harboring Giardia duodenalis, Entamoeba spp., and Blastocystis hominis, in trying to discover possible alterations in their fecal microbiota structure related to the presence of such parasites. METHODOLOGY: Twenty fecal samples were collected from people inhabiting three different localities of Côte d'Ivoire for copromicroscopic analysis and molecular identification of G. duodenalis, Entamoeba spp., and B. hominis. Temporal temperature gradient gel electrophoresis (TTGE) was used to obtain a fingerprint of the overall bacterial community; quantitative polymerase chain reaction (qPCR) was used to define the relative abundances of selected bacterial species/group, and multivariate statistical analyses were employed to correlate all data. RESULTS: Cluster analysis revealed a significant separation of TTGE profiles into four clusters (p < 0.0001), with a marked difference for G. duodenalis-positive samples in relation to the others (p = 5.4×10-6). Interestingly, qPCR data showed how G. duodenalis-positive samples were related to a dysbiotic condition that favors potentially harmful species (such as Escherichia coli), while Entamoeba spp./B. hominis-positive subjects were linked to a eubiotic condition, as shown by a significantly higher Faecalibacterium prausnitzii-Escherichia coli ratio. CONCLUSIONS: This preliminary investigation demonstrates a differential fecal microbiota structure in subjects infected with G. duodenalis or Entamoeba spp./B. hominis, paving the way for using further next-generation DNA technologies to better understand host-parasite-bacteria interactions, aimed at identifying potential indicators of microbiota changes.

Dramatic decrease in prevalence of soil-transmitted helminths and new insights into intestinal protozoa in children living in the Chaco region, Bolivia.

We assessed the prevalence of intestinal parasites among 268 2-12-year-old children living in rural areas, small villages, and semi-urban areas of the Chaco region, south-eastern Bolivia. The overall parasitism was 69%. Only protozoa, helminths, or co-infections were observed in 89.2%, 5.9%, or 4.9% of the positive children, respectively. A significant progressive increase in overall parasite prevalence was found when passing from rural areas to small villages and semi-urban areas. The most commonly found species were Entamoeba coli (38.4%), Giardia intestinalis (37.7%), and Blastocystis spp. (16%). Hymenolepis nana was the most prevalent helminth (5.6%), followed by Ascaris lumbricoides and hookworms (1.5% and 0.4%) evidenced only in rural areas and in villages. Molecular diagnostics identified Blastocystis subtypes 9 and 2, and 5 infections by Entamoeba histolytica and 4 by Entamoeba dispar. The dramatic decrease in prevalence of soil-transmitted helminths with respect to that observed about 20 years ago (> 40%) evidences the success of the preventive chemotherapy intervention implemented in 1986. Health education and improved sanitation should be intensified to control protozoan infections.

Outbreak of Achromobacter xylosoxidans in an Italian Cystic fibrosis center: genome variability, biofilm production, antibiotic resistance, and motility in isolated strains.

Cystic fibrosis (CF) patients have chronic airway infection and frequent exposure to antibiotics, which often leads to the emergence of resistant organisms. Achromobacter xylosoxidans is a new emergent pathogen in CF spectrum. From 2005 to 2010 we had an outbreak in A. xylosoxidans prevalence in our CF center, thus, the present study was aimed at deeply investigating virulence traits of A. xylosoxidans strains isolated from infected CF patients. To this purpose, we assessed A. xylosoxidans genome variability by randomly amplified polymorphic DNA (RAPD), biofilm production, antibiotic resistances, and motility. All A. xylosoxidans strains resulted to be biofilm producers, and were resistant to antibiotics usually employed in CF treatment. Hodge Test showed the ability to produce carbapenemase in some strains. Strains who were resistant to ß-lactamics antibiotics, showed the specific band related to metal ß-lactamase (blaIMP-1), and some of them showed to possess the integron1. Around 81% of A. xylosoxidans strains were motile. Multivariate analysis showed that RAPD profiles were able to predict Forced Expiratory Volume (FEV1%) and biofilm classes. A significant prevalence of strong biofilm producers strains was found in CF patients with severely impaired lung functions (FEV1% class 1). The outbreak we had in our center (prevalence from 8.9 to 16%) could be explained by an enhanced adaptation of A. xylosoxidans in the nosocomial environment, despite of aggressive antibiotic regimens that CF patients usually undergo.

Adherent-invasive Escherichia coli (AIEC) in pediatric Crohn's disease patients: phenotypic and genetic pathogenic features.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC) have been implicated in the ethiopathogenesis of Crohn's disease (CD). In this study, we analyzed a collection of intestinal mucosa-associated E. coli isolates, presenting AIEC phenotypes, isolated from biopsies of CD pediatric patients and non-inflammatory bowel diseases (IBD) controls, in order to investigate their genetic and phenotypic pathogenic features. RESULTS: A total of 616 E. coli isolates from biopsies of four pediatric CD patients and of four non-IBD controls were collected and individually analyzed. For AIEC identification, adherent isolates were assayed for invasiveness, and the capacity of the adhesive-invasive isolates to survive and replicate intracellularly was determined over macrophages J774. In this way we identified 36 AIEC-like isolates. Interestingly, their relative abundance was significantly higher in CD patients (10%; 31/308) than in non-IBD controls (1%; 5/308) (χ2 = 38.96 p < 0.001). Furthermore pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques were applied to analyze the clonality of the 36 AIEC-like isolates. The results obtained allowed us to identify 27 distinct genotypes (22 from CD patients and 5 from non-IBD controls). As for the AIEC prototype strain LF82, all 27 AIEC genotypes presented an aggregative pattern of adherence (AA) that was inhibited by D-mannose, indicating that adhesiveness of AIEC is likely mediated by type 1 pili. PCR analisys was used to investigate presence of virulence genes. The results indicated that among the 27 AIEC isolates, the incidence of genes encoding virulence factors K1 (χ2 = 6.167 P = 0.013), kpsMT II (χ2 = 6.167 P = 0.013), fyuA (χ2 = 6.167 P = 0.013), and ibeA (χ2 = 8.867 P = 0.003) was significantly higher among AIEC strains isolated from CD patients than non-IBD controls. CONCLUSIONS: The identification of AIEC strains in both CD and non-IBD controls, confirmed the "pathobiont" nature of AIEC strains. The finding that AIEC-like isolates were more abundant in CD patients, indicates that a close association of these strains with CD may also exists in pediatric patients.

Bdellovibrio bacteriovorus directly attacks Pseudomonas aeruginosa and Staphylococcus aureus Cystic fibrosis isolates.

Bdellovibrio bacteriovorus is a predator bacterial species found in the environment and within the human gut, able to attack Gram-negative prey. Cystic fibrosis (CF) is a genetic disease which usually presents lung colonization by Pseudomonas aeruginosa or Staphylococcus aureus biofilms. Here, we investigated the predatory behavior of B. bacteriovorus against these two pathogenic species with: (1) broth culture; (2) "static" biofilms; (3) field emission scanning electron microscope (FESEM); (4) "flow" biofilms; (5) zymographic technique. We had the first evidence of B. bacteriovorus survival with a Gram-positive prey, revealing a direct cell-to-cell contact with S. aureus and a new "epibiotic" foraging strategy imaged with FESEM. Mean attaching time of HD100 to S. aureus cells was 185 s, while "static" and "flow" S. aureus biofilms were reduced by 74 (at 24 h) and 46% (at 20 h), respectively. Furthermore, zymograms showed a differential bacteriolytic activity exerted by the B. bacteriovorus lysates on P. aeruginosa and S. aureus. The dual foraging system against Gram-negative (periplasmic) and Gram-positive (epibiotic) prey could suggest the use of B. bacteriovorus as a "living antibiotic" in CF, even if further studies are required to simulate its in vivo predatory behavior.

Higher prevalence and abundance of Bdellovibrio bacteriovorus in the human gut of healthy subjects.

INTRODUCTION: Members of the human intestinal microbiota are key players in maintaining human health. Alterations in the composition of gut microbial community (dysbiosis) have been linked with important human diseases. Understanding the underlying processes that control community structure, including the bacterial interactions within the microbiota itself, is essential. Bdellovibrio bacteriovorus is a gram-negative bacterium that preys other gram-negative species for survival, acting as a population-balancer. It was found in terrestrial/aquatic ecosystems, and in animal intestines, postulating its presence also in the human gut. METHODS: The present study was aimed to evaluate, by end-point PCR and qPCR, the presence of B. bacteriovorus in intestinal and faecal biopsy specimens from 92 paediatric healthy subjects and patients, suffering from Inflammatory Bowel Diseases (IBD), Celiac disease and Cystic fibrosis (CF). RESULTS: i) B. bacteriovorus was present and abundant only in healthy individuals, while it was heavily reduced in patients, as in the case of IBD and Celiac, while in CF patients and relative controls we observed comparable results; ii) B. bacteriovorus seemed to be mucosa-associated, because all IBD and Celiac biopsies (and related controls) were treated with mucus-removing agents, leaving only the mucosa-attached microflora; iii) B. bacteriovorus abundance was district-dependent, with a major preponderance in duodenum, and gradually decreasing up to rectum; iv) B. bacteriovorus levels significantly dropped in disease status, in duodenum and ileum. CONCLUSIONS: Results obtained in this study could represent the first step for new therapeutic strategies aimed to restore a balance in the intestinal ecosystem, utilizing Bdellovibrio as a probiotic.