Periodontopathic bacterial adhesion to different restorative materials used to elevate proximal subgingival margins.

This study compared the periodontopathic bacterial adhesion to four restorative materials used for deep margin elevation at 2, 24, and 48-h after incubation. Discs were produced from four restorative materials: resin modified glass ionomer, glass hybrid, flowable bulk fill resin composite, and bioactive ionic resin. Root dentin was used as control. Specimens were coated with saliva and used to culture a biofilm comprised of three strains of periodontopathic bacteria; Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans. Bacterial adherence was assessed by colony count assay, crystal violet staining, and visualized using confocal laser scanning microscopy. Data were analyzed by two-way ANOVA followed by Tukey's post hoc tests. The adhesion values for the control specimens were significantly higher than for other materials, while those for the flowable bulk fill were significantly lower than for any other material within all evaluation assays. The 2-h incubation period showed the lowest adhesion values regardless of the group. The 48-h adhesion values were higher than the 24-h results in all groups except the flowable bulk fill. Microscopic imaging partially supported the findings of the measurements. In terms of periodontopathic bacterial adhesion, the tested flowable bulk fill may be preferable for subgingival use over other tested materials.

Both LTA and LTB Subunits Are Equally Important to Heat-Labile Enterotoxin (LT)-Enhanced Bacterial Adherence.

There is increasing evidence indicating that the production of heat-labile enterotoxin (LT) enhances bacterial adherence within in vitro and in vivo models. However, which subunit plays the main role, and the precise regulatory mechanisms remain unclear. To further elucidate the contribution of the A subunit of LT (LTA) and the B subunit of LT (LTB) in LT-enhanced bacterial adherence, we generated several LT mutants where their ADP-ribosylation activity or GM1 binding ability was impaired and evaluated their abilities to enhance the two LT-deficient E. coli strains (1836-2 and EcNc) adherence. Our results showed that the two LT-deficient strains, expressing either the native LT or LT derivatives, had a significantly greater number of adhesions to host cells than the parent strains. The adherence abilities of strains expressing the LT mutants were significantly reduced compared with the strains expressing the native LT. Moreover, E. coli 1836-2 and EcNc strains when exogenously supplied with cyclic AMP (cAMP) highly up-regulated the adhesion molecules expression and improved their adherence abilities. Ganglioside GM1, the receptor for LTB subunit, is enriched in lipid rafts. The results showed that deletion of cholesterol from cells also significantly decreased the ability of LT to enhance bacterial adherence. Overall, our data indicated that both subunits are equally responsible for LT-enhanced bacterial adherence, the LTA subunit contributes to this process mainly by increasing bacterial adhesion molecules expression, while LTB subunit mainly by mediating the initial interaction with the GM1 receptors of host cells.

Evaluation of Microbial Adherence on Antibacterial Suture Materials during Intraoral Wound Healing: A Prospective Comparative Study.

AIM: To assess the efficacy of antiseptic-coated silk sutures with triclosan-coated polyglactin 910 suture in reducing bacterial colonization after oral surgical procedures. MATERIALS AND METHODS: The patients who required multiple sutures after surgical procedures in the mandible were the study subjects. The sites of suturing were divided into three groups. Group A - surgical site receiving black-braided silk suture (control group). Group B - surgical site receiving triclosan-coated Polyglactin 910 suture (experimental group). Group C - surgical site receiving antiseptic-coated silk suture (experimental group). Evaluation was done on the 3rd postoperative and 7th postoperative day. Microbial adherence was evaluated by microbiological study. RESULTS: The mean comparison of microbial count between 3rd and 7th post-op day in the three groups shows an increased microbial colonization in the control group when compared with the experimental groups. The combined mean microbial adherence in the three groups showed microbial count in the uncoated silk suture (group A) as 10.35 ± 3.74, triclosan-coated suture (group B) as 6.28 ± 2.17 and iodoform + calendula oil-coated suture (group C) as 7.1 ± 2.02 which is statistically significant (p < 0.05). CONCLUSION: The present research concluded that the pomade-coated silk suture is as efficient as triclosan-coated VICRYL PLUS Polyglactin 910 sutures in reducing the bacterial colonization in intraoral wound healing. CLINICAL SIGNIFICANCE: The pomade (iodoform + calendula oil) may be advocated in the field of oral and maxillofacial surgery for impregnating the suture materials which act as an antiseptic agent and a promoter of wound healing which is easily accessible and also cost-effective.

Association between Brachyspira and irritable bowel syndrome with diarrhoea.

OBJECTIVE: The incidence of IBS increases following enteric infections, suggesting a causative role for microbial imbalance. However, analyses of faecal microbiota have not demonstrated consistent alterations. Here, we used metaproteomics to investigate potential associations between mucus-resident microbiota and IBS symptoms. DESIGN: Mucus samples were prospectively collected from sigmoid colon biopsies from patients with IBS and healthy volunteers, and their microbial protein composition analysed by mass spectrometry. Observations were verified by immunofluorescence, electron microscopy and real-time PCR, further confirmed in a second cohort, and correlated with comprehensive profiling of clinical characteristics and mucosal immune responses. RESULTS: Metaproteomic analysis of colon mucus samples identified peptides from potentially pathogenic Brachyspira species in a subset of patients with IBS. Using multiple diagnostic methods, mucosal Brachyspira colonisation was detected in a total of 19/62 (31%) patients with IBS from two prospective cohorts, versus 0/31 healthy volunteers (p<0.001). The prevalence of Brachyspira colonisation in IBS with diarrhoea (IBS-D) was 40% in both cohorts (p=0.02 and p=0.006 vs controls). Brachyspira attachment to the colonocyte apical membrane was observed in 20% of patients with IBS and associated with accelerated oro-anal transit, mild mucosal inflammation, mast cell activation and alterations of molecular pathways linked to bacterial uptake and ion-fluid homeostasis. Metronidazole treatment paradoxically promoted Brachyspira relocation into goblet cell secretory granules-possibly representing a novel bacterial strategy to evade antibiotics. CONCLUSION: Mucosal Brachyspira colonisation was significantly more common in IBS and associated with distinctive clinical, histological and molecular characteristics. Our observations suggest a role for Brachyspira in the pathogenesis of IBS, particularly IBS-D.

Non-typeable Haemophilus influenzae chronic colonization in chronic obstructive pulmonary disease (COPD).

Haemophilus influenzae is the most common cause of bacterial infection in the lungs of chronic obstructive pulmonary disease (COPD) patients and contributes to episodes of acute exacerbation which are associated with increased hospitalization and mortality. Due to the ability of H. influenzae to adhere to host epithelial cells, initial colonization of the lower airways can progress to a persistent infection and biofilm formation. This is characterized by changes in bacterial behaviour such as reduced cellular metabolism and the production of an obstructive extracellular matrix (ECM). Herein we discuss the multiple mechanisms by which H. influenzae contributes to the pathogenesis of COPD. In particular, mechanisms that facilitate bacterial adherence to host airway epithelial cells, biofilm formation, and microbial persistence through immune system evasion and antibiotic tolerance will be discussed.

Development and assessment of a high-throughput biofilm and biomass testing platform.

OBJECTIVE: To develop and evaluate a simple platform technology for developing static biofilms in a 96-well microtitre plate for various downstream applications. The technology allows monitoring of growth rate, biofilm formation and quantifying biofilm biomass by using crystal violet (CV) and safranin O (SO) staining over seven-day time periods for pathogens including clinical isolates most commonly associated with hard-to-treat wound infections. METHOD: A total of 157 bacteria including Acinetobacter, Enterobacter, Klebsiella, Pseudomonas and Staphylococcus spp. were used in the study. Bacterial growth was measured at 600nm optical density (OD). Biofilm formation was monitored and assessed quantitatively with CV at 570nm and SO staining at 492nm for one-, two-, three- and seven-day incubation periods. RESULTS: Bacterial growth rate and static biofilm biomass in the 96-well plates varied for various strains tested. Both CV and SO staining showed similar results in the biomass, with SO assay displaying more reproducible data throughout the study. Most of the strains were metabolically active even at the seven-day incubation period. Microbial adherences of all bacterial strains on the plastic surface was assessed with CV staining: 28 Acinetobacter, 17 Staphylococcus, 12 Pseudomonas and four Enterobacter strains were strong biofilm producers. Moderate biofilm-producing strains included 27 Staphylococcus, 14 Acinetobacter, eight Pseudomonas and three Enterobacter. Weak biofilm-producing strains included: 33 Staphylococcus, six Enterobacter, two Pseudomonas and one Acinetobacter. Only one Pseudomonas aeruginosa strain did not develop biofilm. CONCLUSION: Our results demonstrate the feasibility of using 96-well microtitre plates as a high-throughput platform for quantitative measurement and assessment of biofilm development over time. Studying microbial adherence or biofilm biomass generated on various surfaces using a high-throughput system could provide valuable information for in vitro testing and developing therapeutics for biofilm infections. Employing the biofilm testing platform described in this study makes it possible to simultaneously develop different biofilms formed by specific pathogens, and study potential association between the quantity of bacterial biomass and strength of a biofilm formed by specific wound pathogens. In addition, the described testing approach could provide an optimal model for standardised and high-throughput screening of candidate antibiofilm therapeutics.

Biocompatible implant mimicking cartilage: A new horizon for reconstructive facial field.

Cartilage is avascular with limited to no regenerative capacity, so its loss could be a challenge for reconstructive surgery. Current treatment options for damaged cartilage are also limited. In this aspect there is a tremendous need to develop an ideal cartilage-mimicking biomaterial that could repair maxillofacial defects. Considering this fact in this study we have prepared twelve silicone-based materials (using Silicone 40, 60, and 80) reinforced with hydroxyapatite, tri-calcium phosphate, and titanium dioxide which itself has proven their efficacy in several studies and able to complement the shortcomings of using silicones. Among the mechanical properties (Young's modulus, tensile strength, percent elongation, and hardness), hardness of Silicone-40 showed similarities with goat ear (P > .05). Silicone peaks have been detected in FTIR. Both AFM morphology and SEM images of the samples confirmed more roughed surfaces. All the materials were nonhemolytic in hemocompatibility tests, but among the twelve materials S2, S3, S5, and S6 showed the least hemolysis. For all tested bacterial strains, adherence was lower on each material than that grown on the plain industrial silicone material which was used as a positive control. S2, S3, S5, and S6 samples were selected as the best based on mechanical characterizations, surface characterizations, in vitro hemocompatibility tests and bacterial adherence activity. So, outcomes of this present study would be promising when developing ideal cartilage-mimicking biocomposites and their emerging applications to treat maxillofacial defects due to cartilage damage.

In vitro evaluation of microbial adhesion to a contraceptive vaginal ring with a new polymer composition.

Objective: The aim of the study was to evaluate in vitro the amount of Candida albicans and Lactobacillus acidophilus adhering to the surface of Ornibel, a contraceptive vaginal ring with a new polymer composition, in comparison with NuvaRing. Methods: Ornibel and NuvaRing were tested for adhesion of microorganisms in vitro. The vaginal rings were aseptically sectioned into 1 cm segments. Suspensions of C. albicans (1-2 × 107 colony-forming units [CFU]/ml) and L. acidophilus (1-2 × 108 CFU/ml) were prepared and incubated either in monoculture or as co-culture with the ring segments. After incubation, adherent C. albicans and L. acidophilus were quantified by plate counting. Results: In monoculture, the measured amount of adhesion of C. albicans on the ring surface was significantly lower with Ornibel compared with NuvaRing (p = 6.77 × 10-5), while the adherence of L. acidophilus did not differ between the two rings. Similarly, when co-incubated with C. albicans, the number of adhering L. acidophilus microorganisms was significantly lower with Ornibel compared with NuvaRing (p = .018) . This reduction also resulted in significantly lower levels of adhering L. acidophilus in co-culture compared with L. acidophilus when incubated alone (p = .003). Conclusion: The study demonstrates that the adherence of C. albicans, as well as that of L. acidophilus when co-cultured with C. albicans, is lower with the Ornibel vaginal ring compared with NuvaRing. These in vitro findings warrant future human trials using molecular techniques to assess the vaginal microbiota composition and to establish whether Ornibel can support vaginal health.

Adherent-invasive Escherichia coli in inflammatory bowel disease.

Intestinal microbiome dysbiosis has been consistently described in patients with IBD. In the last decades, Escherichia coli, and the adherent-invasive E coli (AIEC) pathotype in particular, has been implicated in the pathogenesis of IBD. Since the discovery of AIEC, two decades ago, progress has been made in unravelling these bacteria characteristics and its interaction with the gut immune system. The mechanisms of adhesion of AIEC to intestinal epithelial cells (via FimH and cell adhesion molecule 6) and its ability to escape autophagy when inside macrophages are reviewed here. We also explore the existing data on the prevalence of AIEC in patients with Crohn's disease and UC, and the association between the presence of AIEC and disease location, activity and postoperative recurrence. Finally, we highlight potential therapeutic strategies targeting AIEC colonisation of gut mucosa, including the use of phage therapy, bacteriocins and antiadhesive molecules. These strategies may open new avenues for the prevention and treatment of IBD in the future.

Influenza viral neuraminidase primes bacterial coinfection through TGF-ß-mediated expression of host cell receptors.

Influenza infection predisposes the host to secondary bacterial pneumonia, which is a major cause of mortality during influenza epidemics. The molecular mechanisms underlying the bacterial coinfection remain elusive. Neuraminidase (NA) of influenza A virus (IAV) enhances bacterial adherence and also activates TGF-ß. Because TGF-ß can up-regulate host adhesion molecules such as fibronectin and integrins for bacterial binding, we hypothesized that activated TGF-ß during IAV infection contributes to secondary bacterial infection by up-regulating these host adhesion molecules. Flow cytometric analyses of a human lung epithelial cell line indicated that the expression of fibronectin and α5 integrin was up-regulated after IAV infection or treatment with recombinant NA and was reversed through the inhibition of TGF-ß signaling. IAV-promoted adherence of group A Streptococcus (GAS) and other coinfective pathogens that require fibronectin for binding was prevented significantly by the inhibition of TGF-ß. However, IAV did not promote the adherence of Lactococcus lactis unless this bacterium expressed the fibronectin-binding protein of GAS. Mouse experiments showed that IAV infection enhanced GAS colonization in the lungs of wild-type animals but not in the lungs of mice deficient in TGF-ß signaling. Taken together, these results reveal a previously unrecognized mechanism: IAV NA enhances the expression of cellular adhesins through the activation of TGF-ß, leading to increased bacterial loading in the lungs. Our results suggest that TGF-ß and cellular adhesins may be potential pharmaceutical targets for the prevention of coinfection.

Defining the Mechanical Determinants of Kingella kingae Adherence to Host Cells.

Kingella kingae is an important pathogen in young children and initiates infection by colonizing the posterior pharynx. Adherence to pharyngeal epithelial cells is an important first step in the process of colonization. In the present study, we sought to elucidate the interplay of type IV pili (T4P), a trimeric autotransporter adhesin called Knh, and the polysaccharide capsule in K. kingae adherence to host cells. Using adherence assays performed under shear stress, we observed that a strain expressing only Knh was capable of higher levels of adherence than a strain expressing only T4P. Using atomic force microscopy and transmission electron microscopy (TEM), we established that the capsule had a mean depth of 700 nm and that Knh was approximately 110 nm long. Using cationic ferritin capsule staining and thin-section transmission electron microscopy, we found that when bacteria expressing retractile T4P were in close contact with host cells, the capsule was absent at the point of contact between the bacterium and the host cell membrane. In a T4P retraction-deficient mutant, the capsule depth remained intact and adherence levels were markedly reduced. These results support the following model: T4P make initial contact with the host cell and mediate low-strength adherence. T4P retract, pulling the organism closer to the host cell and displacing the capsule, allowing Knh to be exposed and mediate high-strength, tight adherence to the host cell surface. This report provides the first description of the mechanical displacement of capsule enabling intimate bacterial adherence to host cells.IMPORTANCE Adherence to host cells is an important first step in bacterial colonization and pathogenicity. Kingella kingae has three surface factors that are involved in adherence: type IV pili (T4P), a trimeric autotransporter adhesin called Knh, and a polysaccharide capsule. Our results suggest that T4P mediate initial contact and low-strength adherence to host cells. T4P retraction draws the bacterium closer to the host cell and causes the displacement of capsule. This displacement exposes Knh and allows Knh to mediate high-strength adherence to the host cell. This work provides new insight into the interplay of T4P, a nonpilus adhesin, and a capsule and their effects on bacterial adherence to host cells.

Genomic characterization, phylogenetic analysis, and identification of virulence factors in Aerococcus sanguinicola and Aerococcus urinae strains isolated from infection episodes.

Aerococcus sanguinicola and Aerococcus urinae are emerging pathogens in clinical settings mostly being causative agents of urinary tract infections (UTIs), urogenic sepsis and more seldomly complicated infective endocarditis (IE). Limited knowledge exists concerning the pathogenicity of these two species. Eight clinical A. sanguinicola (isolated from 2009 to 2015) and 40 clinical A. urinae (isolated from 1984 to 2015) strains from episodes of UTIs, bacteremia, and IE were whole-genome sequenced (WGS) to analyze genomic diversity and characterization of virulence genes involved in the bacterial pathogenicity. A. sanguinicola genome sizes were 2.06-2.12 Mb with 47.4-47.6% GC-contents, and 1783-1905 genes were predicted whereof 1170 were core-genes. In case of A. urinae strains, the genome sizes were 1.93-2.44 Mb with 41.6-42.6% GC-contents, and 1708-2256 genes of which 907 were core-genes. Marked differences were observed within A. urinae strains with respect to the average genome sizes, number and sequence identity of core-genes, proteome conservations, phylogenetic analysis, and putative capsular polysaccharide (CPS) loci sequences. Strains of A. sanguinicola showed high degree of homology. Phylogenetic analyses showed the 40 A. urinae strains formed two clusters according to two time periods: 1984-2004 strains and 2010-2015 strains. Genes that were homologs to virulence genes associated with bacterial adhesion and antiphagocytosis were identified by aligning A. sanguinicola and A. urinae pan- and core-genes against Virulence Factors of Bacterial Pathogens (VFDB). Bacterial adherence associated gene homologs were present in genomes of A. sanguinicola (htpB, fbpA, lmb, and ilpA) and A. urinae (htpB, lap, lmb, fbp54, and ilpA). Fifteen and 11-16 CPS gene homologs were identified in genomes of A. sanguinicola and A. urinae strains, respectively. Analysis of these genes identified one type of putative CPS locus within all A. sanguinicola strains. In A. urinae genomes, five different CPS loci types were identified with variations in CPS locus sizes, genetic content, and structural organization. In conclusion, this is the first study dealing with WGS and comparative genomics of clinical A. sanguinicola and A. urinae strains from episodes of UTIs, bacteremia, and IE. Gene homologs associated with antiphagocytosis and bacterial adherence were identified and genetic variability was observed within A. urinae genomes. These findings contribute with important knowledge and basis for future molecular and experimental pathogenicity study of UTIs, bacteremia, and IE causing A. sanguinicola and A. urinae strains.

Comparison of bacterial attachment to platelet bags with and without preconditioning with plasma.

BACKGROUND AND OBJECTIVES: Canadian Blood Services produces apheresis and buffy coat pooled platelet concentrates (PCs) stored in bags produced by two different manufacturers (A and B, respectively), both made of polyvinyl chloride-butyryl trihexyl citrate. This study was aimed at comparing Staphylococcus epidermidis adhesion to the inner surface of both bag types in the presence or absence of plasma factors. MATERIALS AND METHODS: Sets (N = 2-6) of bags type A and B were left non-coated (control) or preconditioned with platelet-rich, platelet-poor or defibrinated plasma (PRP, PPP and DefibPPP, respectively). Each bag was inoculated with a 200-ml S. epidermidis culture adjusted to 0·5 colony-forming units/ml. Bags were incubated under platelet storage conditions for 7 days. After culture removal, bacteria attached to the plastic surface were either dislodged by sonication for bacterial quantification or examined in situ by scanning electron microscopy (SEM). RESULTS: Higher bacterial adhesion was observed to preconditioned PC bags than control containers for both bag types (P < 0·0001). Bacterial attachment to preconditioned bags was confirmed by SEM. Bacteria adhered equally to both types of containers in the presence of PRP, PPP and DefibPPP residues (P > 0·05). By contrast, a significant increase in bacterial adherence was observed to type A bags compared with type B bags in the absence of plasma (P < 0·05) [Correction added on 16 June 2017, after first online publication: this sentence has been corrected]. CONCLUSION: The ability of S. epidermidis to adhere to preconditioned platelet collection bags depends on the presence of plasma factors. Future efforts should be focused on reducing plasma proteins' attachment to platelet storage containers to decrease subsequent bacterial adhesion.

Profiling the Membrane and Glycosaminoglycan-Binding Proteomes of Moraxella catarrhalis.

Moraxella catarrhalis, a Gram-negative bacterium, is an important respiratory pathogen causing acute otitis media and exacerbations of chronic obstructive pulmonary disease. Adhesion of the pathogen to human epithelial cells is mediated via bacterial membrane adhesin proteins. To identify the surface proteome of Moraxella catarrhalis, we applied different membrane protein extraction methods in combination with different proteomic technologies. Proteins from preparations of outer membrane vesicles and from carbonate extractions were analyzed using either a gel-based nano-HPLC-MS/MS technique or 2D-LC-MS/MS. Furthermore, because glycosaminoglycans (GAGs) play an important role for microbial entry into human cells, the GAG-binding membranome of Moraxella catarrhalis was investigated using a glycan-based pull-down approach. By these means, potential vaccine protein candidates that were previously selected by the ANTIGENome technology were confirmed, but importantly also novel proteins were identified as candidates.

The calcium-induced conformation and glycosylation of scavenger-rich cysteine repeat (SRCR) domains of glycoprotein 340 influence the high affinity interaction with antigen I/II homologs.

Oral streptococci adhere to tooth-immobilized glycoprotein 340 (GP340) via the surface protein antigen I/II (AgI/II) and its homologs as the first step in pathogenesis. Studying this interaction using recombinant proteins, we observed that calcium increases the conformational stability of the scavenger-rich cysteine repeat (SRCRs) domains of GP340. Our results also show that AgI/II adheres specifically with nanomolar affinity to the calcium-induced SRCR conformation in an immobilized state and not in solution. This interaction is significantly dependent on the O-linked carbohydrates present on the SRCRs. This study also establishes that a single SRCR domain of GP340 contains the two surfaces to which the apical and C-terminal regions of AgI/II noncompetitively adhere. Compared with the single SRCR domain, the three tandem SRCR domains displayed a collective/cooperative increase in their bacterial adherence and aggregation. The previously described SRCRP2 peptide that was shown to aggregate several oral streptococci displayed limited aggregation and also nonspecific adherence compared to SRCR domains. Finally, we show distinct species-specific adherence/aggregation between Streptococcus mutans AgI/II and Streptococcus gordonii SspB in their interaction with the SRCRs. This study concludes that identification of the metal ion and carbohydrate adherence motifs on both SRCRs and AgI/II homologs could lead to the development of anti-adhesive inhibitors that could deter the adherence of pathogenic oral streptococci and thereby prevent the onset of infections.

Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer.

OBJECTIVE: Colonic mucosa-associated Escherichia coli are increased in Crohn's disease (CD) and colorectal cancer (CRC). They variously haemagglutinate, invade epithelial cell lines, replicate within macrophages, translocate across M (microfold) cells and damage DNA. We investigated genes responsible for these effects and their co-association in colonic mucosal isolates. DESIGN: A fosmid library yielding 968 clones was prepared in E coli EPI300-T1 using DNA from a haemagglutinating CRC isolate, and resulting haemagglutinating clones were 454-pyrosequenced. PCR screening was performed on 281 colonic E coli isolates from inflammatory bowel disease (IBD) (35 patients), CRC (21) and controls (24; sporadic polyps or irritable bowel syndrome). RESULTS: 454-Pyrosequencing of fosmids from the haemagglutinating clones (n=8) identified the afimbrial adhesin afa-1 operon. Transfection of afa-1 into E coli K-12 predictably conferred diffuse adherence plus invasion of HEp-2 and I-407 epithelial cells, and upregulation of vascular endothelial growth factor. E coli expressing afaC were common in CRC (14/21, p=0.0009) and CD (9/14, p=0.005) but not ulcerative colitis (UC; 8/21) compared with controls (4/24). E coli expressing both afaC and lpfA (relevant to M-cell translocation) were common in CD (8/14, p=0.0019) and CRC (14/21, p=0.0001), but not UC (6/21) compared with controls (2/24). E coli expressing both afaC and pks (genotoxic) were common in CRC (11/21, p=0.0015) and UC (8/21, p=0.022), but not CD (4/14) compared with controls (2/24). All isolates expressed dsbA and htrA relevant to intra-macrophage replication, and 242/281 expressed fimH encoding type-1 fimbrial adhesin. CONCLUSIONS: IBD and CRC commonly have colonic mucosal E coli that express genes that confer properties relevant to pathogenesis including M-cell translocation, angiogenesis and genotoxicity.

Phase variation and host immunity against high molecular weight (HMW) adhesins shape population dynamics of nontypeable Haemophilus influenzae within human hosts.

Nontypeable Haemophilus influenzae (NTHi) is a bacterium that resides within the human pharynx. Because NTHi is human-restricted, its long-term survival is dependent upon its ability to successfully colonize new hosts. Adherence to host epithelium, mediated by bacterial adhesins, is one of the first steps in NTHi colonization. NTHi express several adhesins, including the high molecular weight (HMW) adhesins that mediate attachment to the respiratory epithelium where they interact with the host immune system to elicit a strong humoral response. hmwA, which encodes the HMW adhesin, undergoes phase variation mediated by 7-base pair tandem repeats located within its promoter region. Repeat number affects both hmwA transcription and HMW-adhesin production such that as the number of repeats increases, adhesin production decreases. Cells expressing large amounts of HMW adhesins may be critical for the establishment and maintenance of NTHi colonization, but they might also incur greater fitness costs when faced with an adhesin-specific antibody-mediated immune response. We hypothesized that the occurrence of large deletion events within the hmwA repeat region allows NTHi cells to maintain adherence in the presence of antibody-mediated immunity. To study this, we developed a mathematical model, incorporating hmwA phase variation and antibody-mediated immunity, to explore the trade-off between bacterial adherence and immune evasion. The model predicts that antibody levels and avidity, catastrophic loss rates, and population carrying capacity all significantly affected numbers of adherent NTHi cells within a host. These results suggest that the occurrence of large, yet rare, deletion events allows for stable maintenance of a small population of adherent cells in spite of HMW adhesin specific antibody-mediated immunity. These adherent subpopulations may be important for sustaining colonization and/or maintaining transmission.

In vitro model of human mammary gland microbial colonization (MAGIC) demonstrates distinctive cytokine response to imbalanced human milk microbiota.

Despite the established concept of the human mammary gland (MG) as a habitat with its own microbiota, the exact mechanism of MG colonization is still elusive and a well-characterized in vitro model would reinforce studies of the MG microbiota development. We aimed to establish and characterize an in vitro cell model for studying MAmmary Gland mIcrobial Colonization (MAGIC) model. We used the immortalized cell line MCF10A, which expresses the strong polarized phenotype similar to MG ductal epithelium when cultured on a permeable support (Transwell). We analyzed the surface properties of the MAGIC model by gene expression analysis of E-cadherin, tight junction proteins, and mucins and by scanning electron microscopy. To demonstrate the applicability of the model, we tested the adhesion capability of the whole human milk (HM) microbial community and the cellular response of the model when challenged directly with raw HM samples. MCF10A on permeable supports differentiated and formed a tight barrier, by upregulation of CLDN8, MUC1, MUC4, and MUC20 genes. The surface of the model was covered with mucins and morphologically diverse with at least two cell types and two types of microvilli. Cells in the MAGIC model withstood the challenge with heat-treated HM samples and responded differently to the imbalanced HM microbiota by distinctive cytokine response. The microbial profile of the bacteria adhered on the MAGIC model reflected the microbiological profile of the input HM samples. The well-studied MAGIC model could be useful for studies of bacterial attachment to the MG and for in vitro studies of biofilm formation and microbiota development.IMPORTANCEThe MAGIC model may be particularly useful for studies of bacterial attachment to the surface of the mammary ducts and for in vitro studies of biofilm formation and the development of the human mammary gland (MG) microbiota. The model is also useful for immunological studies of the interaction between bacteria and MG cells. We obtained pioneering information on which of the bacteria present in the raw human milk (HM) were able to attach to the epithelium treated directly with raw HM, as well as on the effects of bacteria on the MG epithelial cells. The MAGIC cell model also offers new opportunities for research in other areas of MG physiology, such as the effects of bioactive milk components on microbial colonization of the MG, mastitis prevention, and studies of probiotic development. Since resident MG bacteria may be an important factor in breast cancer development, the MAGIC in vitro tool also offers new opportunities for cancer research.

Presence and Role of the Type 3 Fimbria in the Adherence Capacity of Enterobacter hormaechei subsp. hoffmannii.

Enterobacter hormaechei, one of the species within the Enterobacter cloacae complex, is a relevant agent of healthcare-associated infections. In addition, it has gained relevance because isolates have shown the capacity to resist several antibiotics, particularly carbapenems. However, knowledge regarding colonization and virulence mechanisms of E. hormaechei has not progressed to the same extent as other Enterobacteriaceae species as Escherichia coli or Klebsiella pneumoniae. Here, we describe the presence and role of the type 3 fimbria, a chaperone-usher assembled fimbria, which was first described in Klebsiella spp., and which has been detected in other representatives of the Enterobacteriaceae family. Eight Chilean E. cloacae isolates were examined, and among them, four E. hormaechei isolates were found to produce the type 3 fimbria. These isolates were identified as E. hormaechei subsp. hoffmannii, one of the five subspecies known. A mutant E. hormaechei subsp. hoffmannii strain lacking the mrkA gene, encoding the major structural subunit, displayed a significantly reduced adherence capacity to a plastic surface and to Caco-2 cells, compared to the wild-type strain. This phenotype of reduced adherence capacity was not observed in the mutant strains complemented with the mrkA gene under the control of an inducible promoter. Therefore, these data suggest a role of the type 3 fimbria in the adherence capacity of E. hormaechei subsp. hoffmannii. A screening in E. hormaechei genomes contained in the NCBI RefSeq Assembly database indicated that the overall presence of the type 3 fimbria is uncommon (5.94-7.37%), although genes encoding the structure were detected in representatives of the five E. hormaechei subspecies. Exploration of complete genomes indicates that, in most of the cases, the mrkABCDF locus, encoding the type 3 fimbria, is located in plasmids. Furthermore, sequence types currently found in healthcare-associated infections were found to harbor genes encoding the type 3 fimbria, mainly ST145, ST78, ST118, ST168, ST66, ST93, and ST171. Thus, although the type 3 fimbria is not widespread among the species, it might be a determinant of fitness for a subset of E. hormaechei representatives.

Engagement of α3ß1 and α2ß1 integrins by hypervirulent Streptococcus agalactiae in invasion of polarized enterocytes.

The gut represents an important site of colonization of the commensal bacterium Streptococcus agalactiae (group B Streptococcus or GBS), which can also behave as a deadly pathogen in neonates and adults. Invasion of the intestinal epithelial barrier is likely a crucial step in the pathogenesis of neonatal infections caused by GBS belonging to clonal complex 17 (CC17). We have previously shown that the prototypical CC17 BM110 strain invades polarized enterocyte-like cells through their lateral surfaces using an endocytic pathway. By analyzing the cellular distribution of putative GBS receptors in human enterocyte-like Caco-2 cells, we find here that the alpha 3 (α3) and alpha 2 (α2) integrin subunits are selectively expressed on lateral enterocyte surfaces at equatorial and parabasal levels along the vertical axis of polarized cells, in an area corresponding to GBS entry sites. The α3ß1 and α2ß1 integrins were not readily accessible in fully differentiated Caco-2 monolayers but could be exposed to specific antibodies after weakening of intercellular junctions in calcium-free media. Under these conditions, anti-α3ß1 and anti-α2ß1 antibodies significantly reduced GBS adhesion to and invasion of enterocytes. After endocytosis, α3ß1 and α2ß1 integrins localized to areas of actin remodeling around GBS containing vacuoles. Taken together, these data indicate that GBS can invade enterocytes by binding to α3ß1 and α2ß1 integrins on the lateral membrane of polarized enterocytes, resulting in cytoskeletal remodeling and bacterial internalization. Blocking integrins might represent a viable strategy to prevent GBS invasion of gut epithelial tissues.