Impact of Bacillus subtilis Antibiotic Bacilysin and Campylobacter jejuni Efflux Pumps on Pathogen Survival in Mixed Biofilms.

The foodborne pathogen Campylobacter jejuni is typically found in an agricultural environment; in animals, such as birds, as an intestinal commensal; and also in food products, especially fresh poultry meat. Campylobacter interactions within mixed species biofilms are poorly understood, especially at the microscale. We have recently shown that the beneficial bacterium Bacillus subtilis reduces C. jejuni survival and biofilm formation in coculture by secreting the antibiotic bacillaene. We extend these studies here by providing evidence that besides bacillaene, the antagonistic effect of B. subtilis involves a nonribosomal peptide bacilysin and that the fully functional antagonism depends on the quorum-sensing transcriptional regulator ComA. Using confocal laser scanning microscopy, we also show that secreted antibiotics influence the distribution of C. jejuni and B. subtilis cells in the submerged biofilm and decrease the thickness of the pathogen's biofilm. Furthermore, we demonstrate that genes encoding structural or regulatory proteins of the efflux apparatus system (cmeF and cmeR), respectively, contribute to the survival of C. jejuni during interaction with B. subtilis PS-216. In conclusion, this study demonstrates a strong potential of B. subtilis PS-216 to reduce C. jejuni biofilm growth, which supports the application of the PS-216 strain to pathogen biofilm control. IMPORTANCE Campylobacter jejuni is a prevalent cause of foodborne infections worldwide, while Bacillus subtilis as a potential probiotic represents an alternative strategy to control this alimentary infection. However, only limited literature exists on the specific mechanisms that shape interactions between B. subtilis and C. jejuni in biofilms. This study shows that in the two species biofilms, B. subtilis produces two antibiotics, bacillaene and bacilysin, that inhibit C. jejuni growth. In addition, we provide the first evidence that specific pathogen efflux pumps contribute to the defense against B. subtilis attack. Specifically, the CmeDEF pump acts during the defense against bacilysin, while CmeR-dependent overexpression of CmeABC nullifies the bacillaene attack. The role of specific B. subtilis antibiotics and these polyspecific pumps, known for providing resistance against medically relevant antibiotics, has not been studied during bacterial competition in biofilms before. Hence, this work broadens our understanding of mechanisms that shape antagonisms and defense during probiotic-pathogen interactions.

Bacillaene Mediates the Inhibitory Effect of Bacillus subtilis on Campylobacter jejuni Biofilms.

Biofilms are the predominant bacterial lifestyle and can protect microorganisms from environmental stresses. Multispecies biofilms can affect the survival of enteric pathogens that contaminate food products, and thus, investigating the underlying mechanisms of multispecies biofilms is essential for food safety and human health. In this study, we investigated the ability of the natural isolate Bacillus subtilis PS-216 to restrain Campylobacter jejuni biofilm formation and adhesion to abiotic surfaces as well as to disrupt preestablished C. jejuni biofilms. Using confocal laser scanning microscopy and colony counts, we demonstrate that the presence of B. subtilis PS-216 prevents C. jejuni biofilm formation, decreases growth of the pathogen by 4.2 log10, and disperses 26-h-old preestablished C. jejuni biofilms. Furthermore, the coinoculation of B. subtilis and C. jejuni interferes with the adhesion of C. jejuni to abiotic surfaces, reducing it by 2.4 log10. We also show that contact-independent mechanisms contribute to the inhibitory effect of B. subtilis PS-216 on C. jejuni biofilm. Using B. subtilis mutants in genes coding for nonribosomal peptides and polyketides revealed that bacillaene significantly contributes to the inhibitory effect of B. subtilis PS-216. In summary, we show a strong potential for the use of B. subtilis PS-216 against C. jejuni biofilm formation and adhesion to abiotic surfaces. Our research could bring forward novel applications of B. subtilis in animal production and thus contribute to food safety. IMPORTANCE Campylobacter jejuni is an intestinal commensal in animals (including broiler chickens) but also the most frequent cause of bacterial foodborne infection in humans. This pathogen forms biofilms which enhance survival of C. jejuni in food processing and thus threaten human health. Probiotic bacteria represent a potential alternative in the prevention and control of foodborne infections. The beneficial bacterium Bacillus subtilis has an excellent probiotic potential to reduce C. jejuni in the animal gastrointestinal tract. However, data on the effect of B. subtilis on C. jejuni biofilms are scarce. Our study shows that the B. subtilis natural isolate PS-216 prevents adhesion to the abiotic surfaces and the development of submerged C. jejuni biofilm during coculture and destroys the preestablished C. jejuni biofilm. These insights are important for development of novel applications of B. subtilis that will reduce the use of antibiotics in human and animal health and increase productivity in animal breeding.

Cerebral monitoring during carotid endarterectomy using transcranial cerebral oximetry.

OBJECTIVE: This study aimed to evaluate the outcomes of carotid endarterectomies (CEA) performed using transcranial cerebral oximetry as cerebral monitoringMETHODS: This single-center study included patients treated surgically for significant stenosis of the internal carotid artery (ICA) from January 2012 to December 2017RESULTS: Of the 654 patients, 267 were asymptomatic, and 387 were symptomatic. Eversion CEA was performed in 64.8 %, and patched conventional CEA in 35.2 % of all patients. Totally 11.4 % of all patients had a shunt inserted; all patients with the inserted shunt had the conventional pathed CEA. Severe postoperative complications occurred in 1.5 % of asymptomatic patients and 2.6 % of symptomatic patients. The stroke and death ratio in the shunted group was 2.7 %, and in the non-shunted group, 2.1 % (p = 0.7). We found no significant difference in severe postoperative complications between the shunted and non-shunted group. Further, we found the male gender (p = 0.005), coronary artery disease (p = 0.01), and ongoing smoking (p = 0.003) to be significantly associated with neurological symptoms of the ICA stenosis. We also confirmed current tobacco smoking to be significantly associated with the occurrence of postoperative stroke and death (p = 0.005)CONCLUSION: We found transcranial cerebral oximetry to be reliable in the determination of shunt insertion (Tab. 6, Ref. 26).

Celiac axis compression syndrome - diagnostic and surgical treatment.

INTRODUCTION: Celiac axis compression syndrome is one of the causes of chronic abdominal ischemia. The authors describe their experience with the surgical treatment of Dunbar syndrome in 12 patients. The purpose of the authors was a retrospective analysis of the surgical treatment of celiac axis compression syndrome. METHOD: Between 2007 and 2016, we treated 12 patients for the celiac axis compressive syndrome. Each patient has performed abdominal sonography, CT-angiography, or angiography. The method of choice in these patients was surgical treatment. In 4 patients we resected median arcuate ligament and nerve fibers of celiac axis. In 8 patients we had to resect the stenotic or occluded celiac axis. We reconstructed the blood supply to the celiac axis by the aorto-celiac bypass, using prothetic or venous grafting or patch plastic of celiac axis stenosis. RESULTS: There were no significant complications and deaths in the post - operative period. In one patient signs of chronic abdominal ischemia postoperatively persist. CONCLUSION: Surgery is the method of choice of celiac axis compression syndrome. Indications for surgical reconstruction are symptomatic patients with arterial stenosis over 50%. Proper diagnosis and early surgical treatment are essential for treating chronic visceral ischemia and reducing surgical complications. Key words: celiac axis compression syndrome - chronic abdominal ischemia - resection of ligamentum median arcuate.

LIPOPROTEIN ASSOCIATED PHOSPHOLIPASE A2 AS A MARKER OF VULNERABLE ATHEROSCLEROTIC PLAQUE IN PATIENTS WITH INTERNAL CAROTID ARTERY STENOSIS.

The aim of this study was to compare the concentration of inflammatory vascular markers and morphological structure of atherosclerotic plaque in symptomatic and asymptomatic patients with the stenosis of internal carotid artery (ICA). The research was carried out in 70 patients with hemodynamically significant stenosis of ICA out of which 40 (57%) were asymptomatic patients and 30 (43%) were symptomatic patients, of which 20 patients (66%) have had a stroke, or transient ischemic attack (TIA), 10 patients (33%). All the patients were indicated to carotid endarterectomy as a surgical prevention of stroke. All the patients were taken their blood for biochemical testing (T-Chol, LDL, HDL, TG, Fibrinogen, CRP and specific markers IL-4 and Lp-PLA2) early morning prior to surgery. The highest concentrations of T-Chol, LDL, HDL, CRP and Fibrinogen were measured in symptomatic patients, however, these did not feature a significant difference compared with the group of asymptomatic patients (P>0.05). Significant difference was found in IL-4 (P<0.001) and in Lp-PLA2 (P<0.001). When evaluating concentration of tracked parameters in patients with soft atherosclerotic plaque and patients with calcified atherosclerotic plaque, significant differences were found in these markers: TG (P<0.05), CRP (P<0.01), IL-4 (P<0.001) and Lp-PLA2 (P<0.001). The paper deals with higher concentrations of Lp-PLA2 in patients with a soft atherosclerotic plaque. Higher concentration of Lp-PLA2 and systemic inflammatory markers (CRP, IL-4) could be used along with ultrasonography to detect mainly asymptomatic patients who are in urgent need of surgical or endovascular treatment as a prevention of stroke.

FEATURES OF FORMATION OF COLLATERAL CIRCULATION IN PATIENTS WITH SUBCLAVIAN STEAL SYNDROME.

To date in patients with subclavian steal syndrome diagnosis is only grade of stenosis or localization of occlusion described. Authors recommend to take into account also type of a collateral compensation of cerebral circulation for selection of an optimal treatment The objective of the research was to study the features of formation of collateral circulation in patients with subclavian steal syndrome. The authors described changes in the direction of blood flow in the extracranial vessels of 42 patients with subclavian steal syndrome. Latent subclavian steal syndrome was detected in 26.2% of patients, transient subclavian steal syndrome was found in 54.8% of patients, and a persistent course of the disease was observed in 19.9% of patients. Symptoms of vertebrobasilar insufficiency were detected in 26.6% of patients, and combination of chronic upper extremity ischemia and vertebrobasilar insufficiency was diagnosed in 73.8% of patients. When analyzing the features of collateral circulation in 64.3% of patients the extracranial compensatory mechanism was observed being provided by three main groups of collateral hemodynamic reallocation: the occipito-vertebral hemodynamic mechanism of compensation was detected in 38.1% of cases, the thyroid compensatory mechanism was found in 16.7% of cases, and the brain stem-occipital compensatory mechanism was observed in 9.5% of cases. In 35.7% of patients the intracranial compensatory mechanism was observed being provided by two main groups of collateral hemodynamic reallocation: the vertebro-vertebral compensatory mechanism was found in 21.4% of cases and cerebrobasilar compensatory mechanism was detected in 14.3% of cases. Consideration of the features of collateral circulation in patients with subclavian steal syndrome may serve as a prognostic criterion for selecting an optimal treatment tactics.Each of compensatory mechanisms has its own hemodynamic peculiarities. The occipito- vertebral compensatory mechanism has the most positive influence on the compensationof hemodynamic failure of the vertebrobasilar basin.

Exploring ComQXPA quorum-sensing diversity and biocontrol potential of Bacillus spp. isolates from tomato rhizoplane.

Bacillus subtilis is a widespread and diverse bacterium t exhibits a remarkable intraspecific diversity of the ComQXPA quorum-sensing (QS) system. This manifests in the existence of distinct communication groups (pherotypes) that can efficiently communicate within a group, but not between groups. Similar QS diversity was also found in other bacterial species, and its ecological and evolutionary meaning is still being explored. Here we further address the ComQXPA QS diversity among isolates from the tomato rhizoplane, a natural habitat of B. subtilis, where these bacteria likely exist in their vegetative form. Because this QS system regulates production of anti-pathogenic and biofilm-inducing substances such as surfactins, knowledge on cell-cell communication of this bacterium within rhizoplane is also important from the biocontrol perspective. We confirm the presence of pherotype diversity within B. subtilis strains isolated from a rhizoplane of a single plant. We also show that B. subtilis rhizoplane isolates show a remarkable diversity of surfactin production and potential plant growth promoting traits. Finally, we discover that effects of surfactin deletion on biofilm formation can be strain specific and unexpected in the light of current knowledge on its role it this process.

The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin.

Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.