Bacterial Passage Research Articles

Bacterial transport mediated by micro-nanobubbles in porous media

Determining the role of micro-nanobubbles (MNBs) in controlling the risk posed by pathogens to soil and groundwater during reclaimed water irrigation requires clarification of the mechanism of how MNBs block pathogenic bacteria. In this study, real-time bioluminescence imaging was used to investigate the effects of MNBs on the transport and spatiotemporal distribution of bioluminescent Escherichia coli 652T7 strain in porous media. The presence of MNBs significantly increased the retention of bacteria in the porous media, decreasing the maximum relative effluent concentration (C/C0) by 78 % from 0.97 (without MNBs) to 0.21 (with MNBs). The results suggested that MNBs provided additional sites at the air-water interface (AWI) for bacterial attachment and acted as physical obstacles to reduce bacterial passage. These effects varied with environmental conditions such as solution ionic strength and pore water velocity. The results indicated that MNBs enhanced electrostatic attachment of bacteria at the AWI and their mechanical straining in pores. This study suggests that adding MNBs in pathogen-containing water is an effective measure for increasing filtration efficiency and reducing the risk of pathogenic contamination during agricultural irrigation.

Rational Design of a Robust G-Quadruplex Aptamer as an Inhibitor to Alleviate Listeria monocytogenes Infection.

Listeria monocytogenes (LM) is one of the most invasive foodborne pathogens that cause listeriosis, making it imperative to explore novel inhibiting strategies for alleviating its infection. The adhesion and invasion of LM within host cells are partly orchestrated by an invasin protein internalin A (InlA), which facilitates bacterial passage by interacting with the host cell E-cadherin (E-Cad). Hence, in this work, we proposed an aptamer blocking strategy by binding to the region on InlA that directly mediated E-Cad receptor engagement, thereby alleviating LM infection. An aptamer GA8 with a robust G-quadruplex (G4) structural feature was designed through truncation and base mutation from the original aptamer A8. The molecular docking and dynamics analysis showed that the InlA/aptamer GA8 binding interface was highly overlapping with the natural InlA/E-Cad binding interface, which confirmed that GA8 can tightly and stably bind InlA and block more distinct epitopes on InlA that involved the interaction with E-Cad. On the cellular level, it was confirmed that GA8 effectively blocked LM adhesion with an inhibition rate of 78%. Overall, the robust G4 aptamer-mediated design provides a new direction for the development of inhibitors against other wide-ranging and emerging pathogens.

In vitro evaluation of membranes for regenerative procedures against oral bacteria.

The current literature on guided bone regeneration (GBR) and guided tissue regeneration (GTR) membrane contamination reports that the physicochemical characteristics of these biomaterials might influence affinity to bacteria, which appears to be a major drawback for the clinical outcome of the regenerative procedures. Thus, this study aimed to evaluate, in vitro, a multispecies biofilm adherence and passage of bacteria through different types of commercially available membranes for GTR/GBR. Four types of membranes were tested (n=12): LC) Lumina Coat®; JS) Jason®; BG) Biogide®; and LP) Lumina PTFE®. Aluminum foil (AL) simulated an impermeable barrier and was used as the control. The membranes were adapted to specific apparatus and challenged with a mixed bacterial culture composed of A. actinomycetemcomitans b, S. mutans, S. mitis, and A. israelii. After 2 h or 7 days, bacterial adhesion and passage of bacteria were evaluated through CFU counting, which was analyzed by two-way ANOVA e post hoc Tukey, at a 5% significance level. Representative areas of two membranes of each group were analyzed through scanning electron microscopy (SEM) to assess the morphology and organization of the biofilm over the membrane fibers. LC and LP presented similar values of adhered bacterial cells (p > 0.05), significantly inferior when compared to the other groups, in both time points (p < 0.05). All the tested groups were permeable to bacterial cells, with no significant difference between the trial period of 2 h and 7 days (p > 0.05). SEM analyses demonstrated that adhered bacteria number increased throughout the time points (2 h < 7 days). Commercially available biological membranes demonstrated intense bacterial adherence and passage of bacteria, which increased throughout the trial period.

BACTERIAL TRANSMISSION AND ANTIBIOTIC SENSITIVITY OF CAUSATIVE AGENTS IN PURULENT-SEPTIC COMPLICATIONS IN PATIENTS WITH A SEVERE COURSE OF ACUTE PANCREATITIS

Introduction: Acute pancreatitis has emerged as a leading urgent abdominal condition, with a high mortality rate of up to 70% in cases of purulent-septic complications. The identification of specific causative agents during the infection stage of acute severe pancreatitis is challenging due to the various mechanisms by which pathogens can infiltrate parapancreatic fluid collections. However, early antibiotic prophylaxis is essential in the management of this patient population, considering the potential need for prolonged treatment involving multiple antibiotic rotations. This study aims at analyzing the changes in the microbial species spectrum responsible for purulent-septic complications in acute severe pancreatitis during the course of treatment.&#x0D; Methods: We investigated 174 bacterial cultures obtained from patients diagnosed with acute severe pancreatitis who were treated at the Surgical Department of M.V. Sklifosovsky Poltava Regional Hospital between 2015 and 2018. Among these cultures, 87 were obtained during the initial surgical intervention (39 after initial ultrasound-guided drainage and 38 during the initial open surgical intervention), and 87 were obtained during the course of treatment.&#x0D; Results: Among patients who underwent primary drainage of parapancreatic fluid collections, no aerobic flora was detected in 71.4% of cases, but subsequent bacterial cultures revealed microbial growth in 77.6% of patients. In patients who underwent open surgical intervention, pathological flora was detected in nearly all cases, both during the primary bacterial passage and in subsequent cultures. The species spectrum differed significantly between the primary and repeated bacterial passages. The initial cultures showed a predominance of gram-positive flora (S. epidermidis, Streptococcus spp., Enterococcus spp.), while repeated cultures demonstrated a notable increase in hospital-acquired strains and gram-negative flora (E. coli, Proteus spp., Acinetobacter spp.), along with a nearly three-fold increase in S. aureus. These findings have significant implications for selecting appropriate antibacterial agents throughout the treatment course. Notably, a substantial decrease in the sensitivity of multidrug-resistant strains to reserve antibiotics such as meropenem and trigecycline, by approximately 20%, was observed in subsequent bacterial studies.&#x0D; Conclusion: The microbial spectrum and antibiotic sensitivity of pathogens causing purulent-septic complications in acute severe pancreatitis undergo dynamic changes during the course of treatment. These findings underscore the importance of conducting repeated bacterial cultures and adjusting antibiotic therapy accordingly to optimize treatment outcomes.

Maturation state of colonization sites promotes symbiotic resiliency in the Euprymna scolopes-Vibrio fischeri partnership

BackgroundMany animals and plants acquire their coevolved symbiotic partners shortly post-embryonic development. Thus, during embryogenesis, cellular features must be developed that will promote both symbiont colonization of the appropriate tissues, as well as persistence at those sites. While variation in the degree of maturation occurs in newborn tissues, little is unknown about how this variation influences the establishment and persistence of host-microbe associations.ResultsThe binary symbiosis model, the squid-vibrio (Euprymna scolopes-Vibrio fischeri) system, offers a way to study how an environmental gram-negative bacterium establishes a beneficial, persistent, extracellular colonization of an animal host. Here, we show that bacterial symbionts occupy six different colonization sites in the light-emitting organ of the host that have both distinct morphologies and responses to antibiotic treatment. Vibrio fischeri was most resilient to antibiotic disturbance when contained within the smallest and least mature colonization sites. We show that this variability in crypt development at the time of hatching allows the immature sites to act as a symbiont reservoir that has the potential to reseed the more mature sites in the host organ when they have been cleared by antibiotic treatment. This strategy may produce an ecologically significant resiliency to the association.ConclusionsThe data presented here provide evidence that the evolution of the squid-vibrio association has been selected for a nascent organ with a range of host tissue maturity at the onset of symbiosis. The resulting variation in physical and chemical environments results in a spectrum of host-symbiont interactions, notably, variation in susceptibility to environmental disturbance. This “insurance policy” provides resiliency to the symbiosis during the critical period of its early development. While differences in tissue maturity at birth have been documented in other animals, such as along the infant gut tract of mammals, the impact of this variation on host-microbiome interactions has not been studied. Because a wide variety of symbiosis characters are highly conserved over animal evolution, studies of the squid-vibrio association have the promise of providing insights into basic strategies that ensure successful bacterial passage between hosts in horizontally transmitted symbioses.2h6LgmG9jj4pmTHJMXeiS6Video

Assessment of Intestinal Transcytosis of Neonatal &lt;em&gt;Escherichia coli&lt;/em&gt; Bacteremia Isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate across the gut invade the newborn's bloodstream, causing life-threatening bacteremia. The methodology presented here utilizes polarized intestinal epithelial cells grown on semipermeable inserts to assess the transcytosis of neonatal E. coli bacteremia isolates in vitro. This method uses the established T84 intestinal cell line that has the ability to grow to confluence and form tight junctions and desmosomes. After reaching confluence, mature T84 monolayers develop transepithelial resistance (TEER), which can be quantified using a voltmeter. The TEER values are inversely correlated with the paracellular permeability of extracellular components, including bacteria, across the intestinal monolayer. The transcellular passage of bacteria (transcytosis), on the other hand, does not necessarily alter the TEER measurements. In this model, bacterial passage across the intestinal monolayer is quantified for up to 6 h post-infection, and repeated measurements of TEER are made to monitor the paracellular permeability. In addition, this method facilitates the use of techniques such as immunostaining to study the structural changes in tight junctions and other cell-to-cell adhesion proteins during bacterial transcytosis across the polarized epithelium. The use of this model contributes to the characterization of the mechanisms by which neonatal E. coli transcytose across the intestinal epithelium to produce bacteremia.

Assessment of Intestinal Transcytosis of Neonatal &lt;em&gt;Escherichia coli&lt;/em&gt; Bacteremia Isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate across the gut invade the newborn's bloodstream, causing life-threatening bacteremia. The methodology presented here utilizes polarized intestinal epithelial cells grown on semipermeable inserts to assess the transcytosis of neonatal E. coli bacteremia isolates in vitro. This method uses the established T84 intestinal cell line that has the ability to grow to confluence and form tight junctions and desmosomes. After reaching confluence, mature T84 monolayers develop transepithelial resistance (TEER), which can be quantified using a voltmeter. The TEER values are inversely correlated with the paracellular permeability of extracellular components, including bacteria, across the intestinal monolayer. The transcellular passage of bacteria (transcytosis), on the other hand, does not necessarily alter the TEER measurements. In this model, bacterial passage across the intestinal monolayer is quantified for up to 6 h post-infection, and repeated measurements of TEER are made to monitor the paracellular permeability. In addition, this method facilitates the use of techniques such as immunostaining to study the structural changes in tight junctions and other cell-to-cell adhesion proteins during bacterial transcytosis across the polarized epithelium. The use of this model contributes to the characterization of the mechanisms by which neonatal E. coli transcytose across the intestinal epithelium to produce bacteremia.

Elucidating biofouling over thermal and spatial gradients in seawater membrane distillation in hot climatic conditions

Biofouling is a hurdle of seawater desalination that increases water costs and energy consumption. In membrane distillation (MD), biofouling development is complicated due to the temperature effect that adversely affects microbial growth. Given the high relevance of MD to regions with abundant warm seawater, it is essential to explore the biofouling propensity of microbial communities with higher tolerance to elevated temperature conditions. This study presents a comprehensive analysis of the spatial and temporal biofilm distribution and associated membrane fouling during direct contact MD (DCMD) of the Red Sea water. We found that structure and composition of the biofilm layer played a significant role in the extent of permeate flux decline, and biofilms that built up at 45°C had lower bacterial concentration but higher extracellular polymeric substances (EPS) content as compared to biofilms that formed at 55 °C and 65°C. Pore wetting and bacterial passage to the permeate side were initially observed but slowed down as operating time increased. Intact cells in biofilms dominated over the damaged cells at any tested condition emphasizing the high adaptivity of the Red Sea microbial communities to elevated feed temperatures. A comparison of microbial abundance revealed a difference in bacterial distribution between the feed and biofilm samples. A shift in the biofilm microbial community and colonization of the membrane surface with thermophilic bacteria with the feed temperature increase was observed. The results of this study improve our understanding of biofouling propensity in MD that utilizes temperature-resilient feed waters.

Clarification of large-volume bacterial cultures using a centrifuge-free protocol.

AimsTo provide a reliable, reproducible and centrifuge‐free filtration protocol for clarification of large volumes of bacterial cultures.Methods and ResultsFour experiments were designed to compare different techniques enabling clarification of Escherichia coli cultures using as a benchmark the concentration and quality of bacterial outer membrane vesicles (OMVs). The experiments were designed to examine the performance of different extraction methods on large volume (≥1 L) filtrations of bacterial culture media. Performance parameters included filtration flow rates, sterility testing and characterization of the filtrates by: (i) SDS‐PAGE, (ii) cryogenic transmission electron microscopy, (iii) nanoparticle tracking analysis and (iv) Qubit protein quantification. The experiments revealed that: (i) addition of the filter aid Diatomaceous Earth to the bacterial cultures improved filtration flow rates significantly and eliminated the need for centrifugation prior to filtration; (ii) sterile filtration was successful as no bacterial passage was identified through the membrane filter; (iii) centrifuge‐free filtrates contained an increased amount of OMVs compared to centrifuged filtrates.ConclusionsIn comparison to conventional centrifuge‐based protocols, the clarification method presented has universal applicability for a broad range of microbial extraction procedures, regardless of the volume of culture harvested. Moreover, the decreased amount of OMVs presented in the filtrates following centrifugation step provides an additional argument in favour of a centrifuge‐free approach.Significance and Impact of the StudySterile filtration is a universal method for the clarification of bacterial cultures. Common challenges related to filtration include filter clogging and long processing times, due to limited centrifugation capacity, which can affect product quality. The proposed protocol is likely to ensure a highly effective filtration process and could be a novel approach in improving the filtrate products without the need of centrifugation.

Intestinal Permeability in Children with Functional Gastrointestinal Disorders: The Effects of Diet.

Functional gastrointestinal disorders (FGIDs) are very common and life-impacting in children and young adults, covering 50% of pediatric gastroenterologist consultations. As it is known, FGIDs may be due to alterations in the gut–brain axis, dysbiosis and dysregulation of intestinal barrier, causing leaky gut. This may enhance increased antigen and bacterial passage through a damaged mucosa, worsening the impact of different medical conditions such as FGIDs. Little is known about the role of nutrients in modifying this “barrier disruption”. This narrative review aims to analyze the clinical evidence concerning diet and Intestinal Permeability (IP) in FGIDs in children. We searched the PubMed/Medline library for articles published between January 2000 and November 2021 including children aged 0–18 years old, using keywords related to the topic. Since diet induces changes in the intestinal barrier and microbiota, we aimed at clarifying how it is possible to modify IP in FGIDs by diet modulation, and how this can impact on gastrointestinal symptoms. We found that) is that small changes in eating habits, such as a low-FODMAP diet, an adequate intake of fiber and intestinal microbiota modulation by prebiotics and probiotics, seem to lead to big improvements in quality of life.

Bacterial translocation and microgap formation at a novel conical indexed implant abutment system for single crowns

ObjectivesA conometric concept was recently introduced in which conical implant abutments hold the matching crown copings by friction alone, eliminating the need for cement or screws. The aim of this in vitro study was to assess the presence of microgap formation and bacterial leakage at the Acuris conometric restorative interface of three different implant abutment systems.Material and methodsA total of 75 Acuris samples of three implant-abutment systems (Ankylos, Astra Tech EV, Xive) were subjected to microbiological (n = 60) and scanning electron microscopic (SEM) investigation (n = 15). Bacterial migration into and out of the conical coupling system were analyzed in an anaerobic workstation for 48, 96, 144, and 192 h. Bacterial DNA quantification using qrt-PCR was performed at each time point. The precision of the conometric coupling and internal fit of cemented CAD/CAM crowns on corresponding Acuris TiN copings were determined by means of SEM.Resultsqrt-PCR results failed to demonstrate microbial leakage from or into the Acuris system. SEM analysis revealed minute punctate microgaps at the apical aspect of the conometric junction (2.04 to 2.64 µm), while mean cement gaps of 12 to 145 µm were observed at the crown-coping interface.ConclusionsThe prosthetic morse taper connection of all systems examined does not allow bacterial passage. Marginal integrity and internal luting gap between the ceramic crown and the coping remained within the clinically acceptable limits.Clinical relevanceConometrically seated single crowns provide sufficient sealing efficiency, relocating potential misfits from the crown-abutment interface to the crown-coping interface.

Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn's Disease.

BackgroundThe first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo.MethodsAn in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA.ResultsThere was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls.ConclusionsSublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

Isolation of ultrasmall (filterable) bacteria from patients suffering from ME, and patients and staff of a paediatric hospital

A total of 108 blood samples obtained from 28 male and 80 female patients diagnosed with ME were diluted in sterile, Ringer’s Solution and forced (by suction) through 0.2 µm filters. Of the 28 male samples, 4 yielded filterable bacteria and of the 80 female samples, 18 gave filterable bacteria; as a result, of the total of 124 samples, 22 yielded FB. Filterable (0.4 and 0.2, but not 0.1micron filterable) bacteria were also isolated from the nose throat and skin of paediatric patients and from the throat and skin of staff at an emergency paediatric hospital. The highest percentage of bacterial passage occurred through the largest (0.4 µm) pores. The results show that ultrasmall bacteria occur in ME patients and in paediatric patients and nurses. The potential pathogenic role of such filterable bacteria is briefly discussed.

Relationship between bile salts, bacterial translocation, and duodenal mucosal integrity in functional dyspepsia

Functional dyspepsia (FD) is a complex disorder, in which multiple mechanisms underlie symptom generation, including impaired duodenal barrier function. Moreover, an altered duodenal bile salt pool was recently discovered in patients with FD. We aimed to evaluate the relationship between bile salts, bacterial translocation, and duodenal mucosal permeability in FD. Duodenal biopsies from patients with FD and healthy volunteers (HV) were mounted in Ussing chambers to measure mucosal resistance and bacterial passage in the absence and presence of fluorescein-conjugated Escherichia coli and glyco-ursodeoxycholic acid (GUDCA) exposure. In parallel, duodenal fluid aspirates were collected from patients and bile salts were analyzed. The transepithelial electrical resistance of duodenal biopsies from patients was lower compared with HV (21.4±1.3Ω.cm2 vs. 24.4±1.2Ω.cm2 ; P=.02; N=21). The ratio of glyco-cholic and glyco-chenodeoxycholic acid (GCDCA) to tauro- and GUDCA correlated positively with transepithelial electrical resistance in patients. Glyco-ursodeoxycholic acid slightly altered the mucosal resistance, resulting in similar values between patient and healthy biopsies (22.1±1.0Ω.cm2 vs. 23.0±1.0Ω.cm2 ; P=.5). Bacterial passage after 120minutes was lower for patient than for healthy biopsies (0.0 [0.0-681.8] vs. 1684.0 [0.0-4773.0] E coli units; P=.02). Glyco-ursodeoxycholic acid increased bacterial passage in patient biopsies (102.1 [0.0-733.0] vs. 638.9 [280.6-2124.0] E coli units; P=.009). No correlation was found between mucosal resistance and bacterial passage. Patients with FD displayed decreased duodenal mucosal resistance associated with bile salts, however, not associated with bacterial passage in vitro. In addition, the hydrophilic bile salt glyco-ursodeoxycholic acid abolished differences in mucosal resistance and bacterial passage between patient and control group.

Comparison of antibiotic susceptibility and pathogenicity of wild and serially passaged Streptococcus agalactiae to Nile tilapia (Oreochromis niloticus L.)

The specific objectives of this study were to develop a spontaneously mutated S. agalactiae by serial passage in agar (T1 = control or wild, T2 = 10 days’ serial passage, T3 = 20 days’ serial passage, T4 = 30 days’ serial passage, T5 = 40 days’ serial passage and T6 = 50 days’ serial passage) and to compare the antibiotic susceptibility and pathogenicity of wild and serially passaged S. agalactiae to Nile tilapia (Oreochromis niloticus L.). S. agalactiae in T4 has resulted to increase zone of inhibition (ZOI) to penicillin and ampicillin when compared to T1. As the bacterial passage was increased to 50 days (T6), it resulted to significant decrease on the ZOIs to penicillin and ampicillin. For tetracycline, 40 days’ serial passage (T5) was the most consistent in terms of decreased ZOIs as compared to T1; meanwhile, 30 days’ serial passage (T4) had the most numbered increase in ZOIs. Therefore, T4 and T5 bacterial passages were needed in order to significantly change the ZOIs of S. agalactiae to the antibiotics used in this experiment. In the pathogenicity experiment, except for T4 (20.00%), the wild S. agalactiae (T1 = 30.00%) had lower percent mortality as compared to serially passaged bacterium (T2 = 50.00%, T3 = 50.00%, T5 = 40.00%, T6 = 30.00%). Based on the onset of first mortality, it appeared that 40 (T5) and 50 days (T6) serial passages were less pathogenic as compared to T1. All of the infected fish share the same signs of bacterial infection.

Bacterial Percolation and Sealer Tubular Penetration in a Polymer-based Obturation System Compared with Warm Vertical Condensation Technique: An in Vitro Study.

This study compared bacterial percolation and sealer penetration of a novel obturation technique with the ones of warm vertical condensation technique. A bacterial percolation test was carried out with 80 single rooted human teeth divided into 5 groups; A (n=20): warm vertical condensation and AH-Plus, B (n=20): CPoint with AH-Plus, C (n= 20): CPoint with EndoSequence BC, +ve Control (n=10): teeth with no canal obturation, -ve Control (n=10): teeth with no access cavity. The samples were inoculated with a multispecies bacterial incoulum. Bacterial percolation was evaluated by turbidity. Confocal laser scanning microscopy (CLSM) was used to observe the presence of gaps and voids. Further 48 extracted human mandibular single-canal premolars were used to determine the sealer penetration. Slices of the samples were observed by CLSM to evaluate tubules penetration of the sealer. Kaplan Meyer survival, ANOVA one way and Tuckey HSD test and a Wilcoxon signed-rank test were utilised. A Kaplan-Meier test showed no significant difference overall (P>0.05) between groups A, B and C. At 43 days, the group B showed a significantly inferior ability to prevent bacterial passage (P<0.05). The group C showed a deeper sealer penetration than group A and B with statistically significant differences (P<0.05) for the total penetration (ANOVA one way and Tukey HSD). A Wilcoxon signed-rank test showed statistically significant differences for the penetration in the middle-and apical third of the 3 groups. Based on this in vitro study, the single polymer-cone obturation technique with a resin based- and bioceramic based-sealer behaved similarly to the warm vertical obturation technique in preventing bacterial passage. The bioceramic sealer showed the deepest penetration but did not fully prevent bacterial leakage.

Experimental Listeria\u2013Tetrahymena\u2013Amoeba food chain functioning depends on bacterial virulence traits

BackgroundSome pathogenic bacteria have been developing as a part of terrestrial and aquatic microbial ecosystems. Bacteria are consumed by bacteriovorous protists which are readily consumed by larger organisms. Being natural predators, protozoa are also an instrument for selection of virulence traits in bacteria. Moreover, protozoa serve as a “Trojan horse” that deliver pathogens to the human body. Here, we suggested that carnivorous amoebas feeding on smaller bacteriovorous protists might serve as “Troy” themselves when pathogens are delivered to them with their preys. A dual role might be suggested for protozoa in the development of traits required for bacterial passage along the food chain.ResultsA model food chain was developed. Pathogenic bacteria L. monocytogenes or related saprophytic bacteria L. innocua constituted the base of the food chain, bacteriovorous ciliate Tetrahymena pyriformis was an intermediate consumer, and carnivorous amoeba Amoeba proteus was a consumer of the highest order. The population of A. proteus demonstrated variations in behaviour depending on whether saprophytic or virulent Listeria was used to feed the intermediate consumer, T. pyriformis. Feeding of A. proteus with T. pyriformis that grazed on saprophytic bacteria caused prevalence of pseudopodia-possessing hungry amoebas. Statistically significant prevalence of amoebas with spherical morphology typical for fed amoebas was observed when pathogenic L. monocytogenes were included in the food chain. Moreover, consumption of tetrahymenas fed with saprophytic L. innocua improved growth of A. proteus population while L. monocytogenes-filled tetrahymenas provided negative effect. Both pathogenic and saprophytic bacteria were delivered to A. proteus alive but only L. monocytogenes multiplied within amoebas. Observed differences in A. proteus population behaviour suggested that virulent L. monocytogenes might slow down restoration of A. proteus ability to hunt again and thus restrict the size of A. proteus population. Comparison of isogenic bacterial pairs that did or did not produce the haemolysin listeriolysin O (LLO) suggested a role for LLO in passing L. monocytogenes along the food chain.ConclusionsOur results support the idea of protozoa as a means of pathogen delivery to consumers of a higher order and demonstrated a dual role of protozoa as both a “Trojan horse” and “Troy.”

Integrity of reverse osmosis membrane for removing bacteria: new insight into bacterial passage

Fluorescent microspheres (surrogates for bacteria) allowed identification of the fact that even an intact O-ring seal can allow for some bacterial passage through the reverse osmosis membrane element.

In vitro evaluation of barrier function against oral bacteria of dense and expanded polytetrafluoroethylene (PTFE) membranes for guided bone regeneration.

This study evaluates biofilm formation and barrier function against Streptococcus oralis of nonresorbable polytetrafluoroethylene (PTFE) guided bone regeneration membranes having expanded (e-PTFE) and dense (d-PTFE) microstructure. Three e-PTFE membranes of varying openness, one d-PTFE membrane, and commercially pure titanium discs were evaluated. All e-PTFE membranes consisted of PTFE nodes interconnected by fibrils. The d-PTFE membrane was fibril-free, with large evenly spaced indentations. The surfaces were challenged with S. oralis and incubated statically for 2-48h. Bacterial colonization, viability, and penetration were evaluated. S. oralis numbers increased over time on all surfaces, as observed using scanning electron microscopy, while cell viability decreased, as measured by colony forming unit (CFU) counting. At 24h and 48h, biofilms on d-PTFE were more mature and thicker (tower formations) than on e-PTFE, where fewer layers of cells were distributed mainly horizontally. Biofilms accumulated preferentially within d-PTFE membrane indentations. At 48h, greater biofilm biomass and number of viable S. oralis were found on d-PTFE compared to e-PTFE membranes. All membranes were impermeable to S. oralis cells. All PTFE membranes were effective barriers against bacterial passage in vitro. However, d-PTFE favored S. oralis biofilm formation.

Vasoactive Intestinal Polypeptide and Mast Cells Regulate Increased Passage of Colonic Bacteria in Patients With Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is associated with intestinal dysbiosis and symptoms of IBS develop following gastroenteritis. We aimed to study the passage of live bacteria through the colonic epithelium, and determine the role of mast cells (MCs) and vasoactive intestinal polypeptide (VIP) in barrier regulation in IBS and healthy individuals. Colon biopsies from 32 women with IBS and 15 age-matched healthy women (controls) were mounted in Ussing chambers; we measured numbers of fluorescently labeled Escherichia coli HS and Salmonella typhimurium that passed through from the mucosal side to the serosal side of the tissue. Some biopsies were exposed to agents that block the VIP receptors (VPAC1 and VPAC2) or MCs. Levels of VIP and tryptase were measured in plasma and biopsy lysates. Number of MCs and MCs that express VIP or VIP receptors were quantified by immunofluorescence. Biopsies from an additional 5 patients with IBS and 4 controls were mounted in chambers and Salmonella were added; we studied passage routes through the epithelium by transmission electron microscopy and expression of tight junctions by confocal microscopy. In colon biopsies from patients with IBS, larger numbers of E coli HS and Styphimurium passed through the epithelium than in biopsies from controls (P < .0005). In transmission electron microscopy analyses, bacteria were found to cross the epithelium via only the transcellular route. Bacterial passage was reduced in biopsies from patients with IBS and controls after addition of antibodies against VPACs or ketotifen, which inhibits MCs. Plasma samples from patients with IBS had higher levels of VIP than plasma samples from controls. Biopsies from patients with IBS had higher levels of tryptase, larger numbers of MCs, and a higher percentage of MCs that express VPAC1 than biopsies from controls. In biopsies from patients with IBS, addition of Salmonella significantly reduced levels of occludin; subsequent addition of ketotifen significantly reversed this effect. We found that colonic epithelium tissues from patients with IBS have increased translocation of commensal and pathogenic live bacteria compared with controls. The mechanisms of increased translocation include MCs and VIP.