Listeria monocytogenes is a human pathogen, ubiquitous in the environment, and can grow and survive under a wide range of environmental conditions. It contaminates foods via raw materials or food-processing environments. However, the current knowledge of its ecology and, in particular, the mode of environmental survival and transmission of this intracellular pathogen remains limited. Research has shown that several intracellular pathogens are able to survive or replicate within free-living amoebae. To examine the viability of L. monocytogenes in interaction with Acanthamoeba spp., bacteria were co-cultured with three freshly isolated amoebae, namely Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba lenticulata. The survival of bacteria and amoebae was determined using culture techniques and microscopy. Under the experimental conditions used, all amoebae were able to eliminate bacteria irrespective of the hly gene. Bacteria did not survive or replicate within amoeba cells. However, extra-amoebic bacteria grew saprophytically on materials released from amoebae, which may play an important role in the survival of bacteria under extreme environmental conditions.

Listeria monocytogenes is a ubiquitous bacterium capable of infecting humans, particularly pregnant women and immunocompromised individuals. Although the intracellular invasion and pathogenesis of listeriosis in mammalian tissues has been well studied, little is known about the ecology of L. monocytogenes, and in particular the environmental reservoir for this bacterium has not been identified. This study used short-term co-culture at 15, 22 and 37 6C to examine the interaction of L. monocytogenes strains with Acanthamoeba polyphaga ACO12. Survival of L. monocytogenes cells phagocytosed by monolayers of trophozoites was assessed by culture techniques and microscopy. A. polyphaga trophozoites eliminated bacterial cells within a few hours post-phagocytosis, irrespective of the incubation temperature used. Wild-type L. monocytogenes and a phenotypic listeriolysin O mutant were unable to either multiply or survive within trophozoites. By contrast, Salmonella enterica serovar Typhimurium C5 cells used as controls were able to survive and multiply within A. polyphaga trophozoites. The data presented indicate that A. polyphaga ACO12 is unlikely to harbour L. monocytogenes, or act as an environmental reservoir for this bacterium. INTRODUCTIONListeria monocytogenes is a well-known Gram-positive, opportunistic, intracellular bacterial pathogen capable of infecting humans and animals (Allerberger, 2003; Vázquez-Boland et al., 2001). L. monocytogenes is ubiquitous in the environment and can survive and grow in a wide range of environmental conditions (Roberts & Wiedmann, 2003; Vázquez-Boland et al., 2001). This versatility may explain the ability of L. monocytogenes to contaminate a variety of foods and food-processing environments (Roberts & Wiedmann, 2003). Foods contaminated with this bacterium represent a serious health concern in developed countries, as food-borne outbreaks of listeriosis are characterized by a high case mortality rate (20-30 %) (Herd & Kocks, 2001;Roberts & Wiedmann, 2003; Vázquez-Boland et al., 2001;Wing & Gregory, 2002).The mechanism of intracellular invasion, dissemination and survival of L. monocytogenes in mammalian cells has been well characterized in vitro (Cossart, 2002;Cossart & Sansonetti, 2004). However, comparatively little is known about the ecology of this intracellular pathogen in natural habitat, and unlike other intracellular pathogenic bacteria, no host reservoir has been identified (Zhou et al., 2007). If indeed L. monocytogenes lacks a host reservoir, it is intriguing that this pathogen has maintained an ability to invade and parasitize mammalian cells in the absence of selection for the genes necessary for internalization by host cells, escape from the host phagolysosome to the cytoplasmic compartment, and infection of neighbouring host cells.Studies of interactions between amoebae and various intracellular bacterial pathogens have suggested that freeliving amoebae are able to harbour bacterial pathogens (Harb et al., 2000;Molmeret et al., 2005). For example, Salmonella enterica...

Klebsiella pneumoniae is one of the major causes of nosocomial infections worldwide which can cause several diseases in children and adults. The globally dissemination of hyper-virulent strains of K. pneumoniae and the emergence of antibioticsresistant isolates of this pathogen narrows down the treatment options and has renewed interest in its vaccines. Vaccine candidates of Klebsiella pneumoniae have not been adequately protective, safe and globally available yet. In K. pneumoniae infection, it is well known that B cells that induce robust humoral immunity are necessary for the host complete protection. Identifying the B cell epitopes of antigens is valuable to design novel vaccine candidates. In the present study using immunoinformatics approaches we found B cell epitopes of four K. pneumoniae type 1 fimbriae antigens namely FimA, FimF, FimG, and FimH. Linear and conformational B cell epitopes of each antigen were predicted using different programs. Subsequently, many bioinformatics assays were applied to choose the best epitopes including prediction antigenicity, toxicity, human similarity and investigation on experimental records. These assays resulted in final four epitopes (each for one Fim protein). These final epitopes were modeled and their physiochemical properties were estimated to be used as potential vaccine candidates. Altogether, we found four B cell epitopes of K. pneumoniae Fim antigens that are immunogen, antigenic, not similar to human peptides, not allergen and not toxic. Also, they have suitable physiochemical properties to administrate as vaccine, although their complete efficacy should be also shown in vitro and in vivo.

Intra-cellular pathogen, Listeria monocytogenes, is capable of invasion and survival within mammalian cells. However, Acanthamoeba polyphaga trophozoites phagocytose and rapidly degrade Listeria cells. In order to provide more information on amoeba phagocytosis and killing mechanisms, this study used several inhibitor agents known to affect the phagocytosis and killing of bacteria by eukaryotes. Amoebae were pre-treated with mannose, cytochalasin D, wortmannin, suramin, ammonium chloride, bafilomycin A and monensin followed by co-culture with bacteria. Phagocytosis and killing of bacterial cells by amoeba trophozoites was assessed using plate counting methods and microscopy. The data presented indicates that actin polymerisation and cytoskeletal rearrangement are involved in phagocytosis of L. monocytogenes cells by A. polyphaga trophozoites. Further, both phagosomal acidification and phagosome-lysosome fusion are involved in killing and degradation of L. monocytogenes cells by A. polyphaga. However, the mannose-binding protein receptor does not play an important role in uptake of bacteria by amoeba trophozoites. In conclusion, this data reveals the similar principles of molecular mechanisms used by different types of eukaryotes in uptake and killing of bacteria.

Background:Pseudomonas aeruginosa is an opportunistic pathogen associated with nosocomial infections. The emergence and dissemination of metallo-beta-lactamases (MBLs) has contributed to the high rate of resistance among P. aeruginosa isolates.Objectives:The purpose of this study was to describe the prevalence and the clonal dissemination of MBL- producing P. aeruginosa isolates collected from major hospitals in Kermanshah.Materials and Methods:Antibiotic susceptibility testing was performed using the minimal inhibitory concentrations. The MBLs were investigated using the Double-Disk Synergy Test (DDST) and Polymerase Chain Reaction. Molecular typing was performed by Pulsed-Field Gel Electrophoresis (PFGE).Results:Of the 60 P. aeruginosa isolates included in this study, 30 (50%) were resistant to Gentamicin, 38 (63.3%) to Piperacillin, 42 (70%) to Ceftazidime, and 45 (75%) to Cefepime. Twenty-nine (48.3%) isolates were MBL producers in the DDST test. Five (8.3%) isolates were positive for the VIM gene. PFGE analysis among the MBL producers revealed 12 distinct clonal patterns.Conclusions:The inter- and intra-hospital dissemination of resistant clones is a matter of concern and is an indicator of the level of the improvement and surveillance of standard hygiene, particularly disinfection and hand washing before and after contact with patients. Given the emergence of MBL-producing strains, surveillance has become an important procedure to control the transmission of resistant strains.

BACKGROUNDNeonatal sepsis is a disease process, which represents the consequences of systemic response to bacteria entering the blood stream during the first 28 days of life. Platelet count (PLT) and indices, including mean platelet volume (MPV), platelet distribution width (PDW) and Platelet larger cell ratio (P-LCR) are considered as markers of production rate and platelet activation. They are readily available via blood tests. However, their prognostic value in neonatal sepsis has not been fully clarified and several studies reported contradictory results. This study evaluated the changes of platelet count and indices in neonatal sepsis. METHODSThis study was performed on 20 neonates with culture proven sepsis and 20 neonates with the similar age and weight but without any infectious disease served as the control group from February 2013 to June 2017 in the west of Iran. PLT, MPV, PDW, P-LCR and other infection markers (white blood cell count [WBC], erythrocyte sedimentation rate [ESR], and neutrophil percentage were also determined. This data was compared between patients and control group. RESULTSIn sepsis group, MPV, PDW and P-LCR were significantly increased (p= 0.024, p= 0.006 and p= 0.005 respectively), but WBC, Neutrophil percentage and PLT were not significantly different compared to controls. There was no significant difference in the platelet count and indices between Gram-negative and Gram-positive sepsis. CONCLUSIONSDiagnostic value of platelet indices is higher than that of WBC and neutrophil percentage. Therefore, platelet indices as inexpensive and easily available tests can be routinely performed for all neonates suspected to be in sepsis.

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In spite of numerous studies on vaccination for various species of Leishmania, research on the development of an effective vaccine for L. tropica is very scarce. In silico epitope prediction is a new way to survey the best vaccine candidates. Here, we predicted the best epitopes of six L. tropica antigens with vaccine capability against this pathogen, using highly frequent HLA-I alleles. Based on the frequent HLA alleles, the protein sequences were screened individually using four different MHC prediction applications, namely SYFPEITHI, ProPredI, BIMAS, and IEDB. Several in silico assays including clustering, human similarity exclusion, epitope conservancy prediction, investigating in experimental records, immunogenicity prediction, and prediction of population coverage were performed to narrow the results and to find the best epitopes. The selected epitopes and their restricted HLA-I alleles were docked and the final epitopes with the lowest binding energy (the highest binding affinity) were chosen. Finally, the stability and the binding properties of the best epitope-HLA-I combinations were analyzed using molecular dynamics simulation studies. We found ten potential peptides with strong binding affinity to highly frequent HLA-I alleles that can be further evaluated as vaccine targets against L. tropica.