Coccoid Forms Research Articles

Anti-biofilm activity of A22 ((S-3,4-dichlorobenzyl) isothiourea hydrochloride) against Pseudomonas aeruginosa: Influence on biofilm formation, motility and bioadhesion

Bacterial biofilms are involved in various medical infections and for this reason it is of great importance understanding adhesion mechanisms of involved microorganisms is essential to develop new strategies of prevention and control. Different approaches have been used for preventing biofilm related infections in health care settings, such as use of surface coatings agents in medical implants. In this context, is necessary to explore new compounds with anti-biofilm activity. Thus, this study evaluated for the first time the action of A22 against biofilms of Pseudomonas aeruginosa PAO1 strain and multi-resistant clinical isolates on biotic and abiotic surfaces. A22 acts as inhibitor of the MreB protein of the bacterial cell wall, causing the rods to change shape to the coccoid form. In this work, A22 at subinhibitory concentrations was able to prevent biofilm formation, and atomic force microscopy images showed that A22 was highly effective in inhibiting adhesion on polyethylene surfaces. Pseudomonas aeruginosa PAO1 exhibited a strong ability to adhere to HeLa cells, and A22 inhibited the aggregation after 4 h of exposure. Swarming and twitching motilities were significantly altered by A22 at subinhibitory concentrations. Thus, by changing the shape of the bacterial cell, many properties can be affected, such as motility, surface adhesion and biofilm formation. This work presents A22 as a promising novel antibacterial or surface coating agent of medical materials.

Genomic variations leading to alterations in cell morphology of Campylobacter spp.

Campylobacter jejuni, the most common cause of bacterial diarrhoeal disease, is normally helical. However, it can also adopt straight rod, elongated helical and coccoid forms. Studying how helical morphology is generated, and how it switches between its different forms, is an important objective for understanding this pathogen. Here, we aimed to determine the genetic factors involved in generating the helical shape of Campylobacter. A C. jejuni transposon (Tn) mutant library was screened for non-helical mutants with inconsistent results. Whole genome sequence variation and morphological trends within this Tn library, and in various C. jejuni wild type strains, were compared and correlated to detect genomic elements associated with helical and rod morphologies. All rod-shaped C. jejuni Tn mutants and all rod-shaped laboratory, clinical and environmental C. jejuni and Campylobacter coli contained genetic changes within the pgp1 or pgp2 genes, which encode peptidoglycan modifying enzymes. We therefore confirm the importance of Pgp1 and Pgp2 in the maintenance of helical shape and extended this to a wide range of C. jejuni and C. coli isolates. Genome sequence analysis revealed variation in the sequence and length of homopolymeric tracts found within these genes, providing a potential mechanism of phase variation of cell shape.

Leukocyte activation receptor CD69 and ligands: Immunoregulatory role in inflammatory processes

Helicobacter pylori (H. pylori) has been extensively implicated in the etiology and pathogenesis of gastric ulcers and carcinomas, which has necessitated its efficient and cost-effective identification in gastric biopsy samples. We have sought to compare various staining methods, namely, Hematoxylin and Eosin (H&E), Giemsa, and Modified Toluidine Blue (MTB), with immunohistochemistry (IHC; gold standard) in terms of efficacy, staining costs, and duration of performing the stains in settings with limited resources.Gastric biopsy specimens of 50 patients who presented with gastritis symptoms were stained with H&E, Giemsa, MTB, and IHC. The sensitivity, specificity, positive/negative predictive values, and the receiver operating characteristic curve were calculated for each stain.In all, 32 cases of 50 were positive for H. pylori on IHC. The specificity for both H&E and Giemsa was 88.8%, whereas it was lower for MTB (83.3%). The sensitivity of H&E was much lower (46.8%) compared with the other stains (90.6% Giemsa, 93.7% MTB). The most inexpensive and time-consuming stain was H&E followed by MTB and Giemsa.When H&E is used alone for H. pylori detection, a significant number of cases may be overlooked, especially mild inflammatory cases and when coccoid forms of the bacteria are present. This study proposes the use of either Giemsa or MTB as reliable alternatives for IHC in resource-limited settings to combat the high prevalence of H. pylori in the developing world.

Biodiversity of green algae covering artificial hard substrate surfaces in a suburban environment: a case study using molecular approaches.

In Middle European suburban environments green algae often cover open surfaces of artificial hard substrates. Microscopy reveals the Apatococcus/Desmococcus morphotype predominant over smaller coccoid forms. Adverse conditions such as limited water availability connected with high PAR and UV irradiance may narrow the algal diversity to a few specialists in these subaerial habitats. We used rRNA gene cloning/sequencing from both DNA extracts of the biofilms without culturing as well as cultures, for the unambiguous determination of the algal composition and to assess the algal diversity more comprehensively. The culture independent approach revealed mainly just two genera (Apatococcus, Trebouxia) for all study sites and five molecular operational taxonomic units (OTUs) for a particular study site, which based on microscopic observation was the one with the highest morphological diversity. The culture approach, however, revealed seven additional OTUs from five genera (Chloroidium, Coccomyxa, Coenochloris, Pabia, Klebsormidium) and an unidentified trebouxiophyte lineage for that same site; only two OTUs were shared by both approaches. Two OTUs or species were recovered for which references have been isolated only from Antarctica so far. However, the internal transcribed spacer (ITS) sequence differences among them supported they are representing distinct populations of the same species. Within Apatococcus five clearly distinct groups of ITS sequences, each putatively representing a distinct species, were recovered with three or four such ITS types co-occurring at the same study site. Except for the streptophyte Klebsormidium only members of Trebouxiophyceae were detected suggesting these algae may be particularly well-adapted to subaerial habitats.

Effects of ozone and chlorine disinfection on VBNC Helicobacter pylori by molecular techniques and FESEM images

ABSTRACTHelicobacter pylori is a pathogen bacteria associated with chronic gastritis, peptic ulceration, and gastric carcinoma. H. pylori has a spiral morphology, which under certain conditions of stress becomes a coccoid form. This type of morphology has been linked to a viable but non-culturable (VBNC) state, which is thought to allow its persistence in the environment. Membrane damage in VBNC H. pylori in water as a mechanism for inactivation using ozone (O3) and chlorine disinfection has not been reported in the literature. In this paper, disinfection assays with ozone and chlorine were conducted to evaluate their effects on VBNC H. pylori cells. The use of fluorescent dyes such as propidium monoazide (PMA) coupled with quantitative real-time polymerase chain reactions produced results necessary to assess the viability of the microorganism and demonstrate the effect of each disinfectant on the bacterial count. Applying ozone showed a 5-log bacterial reduction using a disinfectant concentration and exposure time (CT) of 4 mg min/L. Chlorine disinfection for the same 5-log reduction required a higher CT value. Field emission scanning electron microscope images of ozone-treated VBNC H. pylori also showed severe cell damage. The use of PMA revealed that chlorine produced physical damage in the membrane in addition to the known inhibiting effect on cell enzymatic processes. These findings are important for the detection and control of VBNC H. pylori cells in drinking water systems.

Antibacterial, cyto and genotoxic activities of A22 compound ((S-3, 4 -dichlorobenzyl) isothiourea hydrochloride)

The A22 is a chemical compound that acts as a reversible inhibitor of a bacterial cell wall protein MreB leading the rods to the coccoid form. Thus, by changing the bacterial form, many properties can be affected, as the acquisition of nutrients, cell division, the clamping surfaces, motility and pathogenesis. Infections caused by strains of Pseudomonas aeruginosa have great clinical importance because these microorganisms can include more than one resistance mechanism acting together, limiting treatment options. Thus, it is important to investigate the action of A22 against P. aeruginosa, once there are urgent needs for new antimicrobial compounds for increase the arsenal therapeutic to treat diseases caused by this microrganism. Therefore, this study investigated for the first time the antimicrobial activity of A22 against seve standards strains of Gram negative microorganisms and twenty-eight clinical isolates of P. aeruginosa. This study performed an additional investigation to analyze the cyto and genotoxic potential effects from A22 on human peripheral blood mononuclear cells (PBMCs). The antibacterial activity of A22 was studied by broth microdilution method and time-kill assay. The cytotoxicity was evaluated by MTT assay at 24, 48 and 72 h of exposure to A22 and the genotoxicity was evaluated by the Comet assay. The susceptibility tests showed A22 has a relevant antibacterial activity against P. aeruginosa, including multidrug-resistant (MDR) clinical isolates. The A22 treatment not showed genotoxic effects against PBMCs in almost all concentrations tested at 24 and 48 h of exposure. Only for concentration of 32 μg/mL (highest tested) the damage index was significantly higher in all moments. The MTT assay demonstrated that A22 was able to maintain cell viability in all exposure times. In summary, the A22 demonstrated important anti-Pseudomonas activity and showed no cyto and genotoxic significant effect. These results need to be considered in future in vitro and in vivo studies in order to introduce the A22 as a possible therapeutic option.

English

Helicobactor pylori (H. pylori) has been strongly associated with gastritis, peptic ulcer and is linked to an increased risk of gastric cancer. The cytotoxin-associated gene product (cagA) and the vacuolating cytotoxin (vacA) have been implicated as two major virulence factors of H. pylori. Since there is an increasing evidence that genetic variability of H. pylori may have clinical importance, we aimed to evaluate different vacA genotypes and reveal its relationship with endoscopic and transmission electron microscopy (TEM) findings among H. pylori infected Egyptian patients. Forty H. pylori infected patients possessing vacA gene who underwent upper endoscopic examination were considered to be infected with H. pylori when rapid urease test and detection of 16S rRNA in gastric biopsy recorded positive. Both vacA and cagA genotypes were detected by polymerase chain reaction (PCR). The TEM was performed to assess the ultra-structure of the gastric mucosa. Four vacA genotypes were identified, the most prominent was the s2/m2 allele combination (52.5%) followed by s1/m1 (27.5%), s1/m2 (17.5%) and s2/m1 genotype was found just in one H. pylori strain (2.5%). There were significant correlations between vacA s2/m2 and gastritis (65.2%), and vacA s1/m1 and peptic ulceration (57%). The cagA gene was associated with 38% of vacA genotypes and 60% of which were significantly associated with vacA s1/m1 genotype with the development of severe gastritis reaching up to gastric ulcer. The TEM revealed H. pylori spiral and coccoid forms, cytoplasmic vacuolar degeneration caused by vacA, swollen mitochondria and dilated rough endoplasmic reticulum. In Egypt where prevalence of H. pylori infection is high, genotyping of H. pylori virulence factors can help to predict patients who are at a high risk of related gastroduodenal diseases. Although H. pylori with vacA s2/m2 genotype is mostly related to low level of virulent strains yet, significant crosstalk between H. pylori strains harboring both vacA s1/m1 and cagA gene provides crucial insights into virulence of high level. Key words: Helicobactor pylori, vacA genotyping, cagA, gastritis, peptic ulcer.

Intrinsic characteristics of Min proteins on the cell division of Helicobacter pylori.

Helicobacter pylori divides in the human stomach resulting in persistent infections and causing various disorders. Bacterial cell division is precisely coordinated by many molecules, including FtsZ and Min proteins. However, the role of Min proteins in H. pylori division is poorly understood. We investigated the functional characteristics of Min proteins in wild-type HPK5 and five HPK5-derivative mutants using morphological and genetic approaches. All mutants showed a filamentous shape. However, the bacterial cell growth and viability of three single-gene mutants (minC, minD, minE) were similar to that of the wild-type. The coccoid form number was lowest in the minE-disruptant, indicating that MinE contributes to the coccoid form conversion during the stationary phase. Immunofluorescence microscopic observations showed that FtsZ was dispersedly distributed throughout the bacterial cell irrespective of nucleoid position in only minD-disruptants, indicating that MinD is involved in the nucleoid occlusion system. A chase assay demonstrated that MinC loss suppressed FtsZ-degradation, indicating that FtsZ degrades in a MinC-dependent manner. Molecular interactions between FtsZ and Min proteins were confirmed by immunoprecipitation (IP)-western blotting (WB), suggesting the functional cooperation of these molecules during bacterial cell division. This study describes the intrinsic characteristics of Min proteins and provides new insights into H. pylori cell division.

Galectin-3 Plays an Important Role in Innate Immunity to Gastric Infection by Helicobacter pylori.

We studied the role of galectin-3 (Gal3) in gastric infection by Helicobacter pylori We first demonstrated that Gal3 was selectively expressed by gastric surface epithelial cells and abundantly secreted into the surface mucus layer. We next inoculated H. pylori Sydney strain 1 into wild-type (WT) and Gal3-deficient mice using a stomach tube. At 2 weeks postinoculation, the bacterial cells were mostly trapped within the surface mucus layer in WT mice. In sharp contrast, they infiltrated deep into the gastric glands in Gal3-deficient mice. Bacterial loads in the gastric tissues were also much higher in Gal3-deficient mice than in WT mice. At 6 months postinoculation,H. pylori had successfully colonized within the gastric glands of both WT and Gal3-deficient mice, although the bacterial loads were still higher in the latter. Furthermore, large lymphoid clusters mostly consisting of B cells were frequently observed in the gastric submucosa of Gal3-deficient mice.In vitro, peritoneal macrophages from Gal3-deficient mice were inefficient in killing engulfed H. pylori Furthermore, recombinant Gal3 not only induced rapid aggregation of H. pylori but also exerted a potent bactericidal effect on H. pylori as revealed by propidium iodide uptake and a morphological shift from spiral to coccoid form. However, a minor fraction of bacterial cells, probably transient phase variants of Gal3-binding sugar moieties, escaped killing by Gal3. Collectively, our data demonstrate that Gal3 plays an important role in innate immunity to infection and colonization of H. pylori.

Helicobacter pullorum induces nitric oxide release in murine macrophages that promotes phagocytosis and killing.

Helicobacter pullorum is an avian enterohepatic species that, more recently, has also been found as a naturally acquired infection in mice and rats, and isolated from patients with gastrointestinal and hepatobiliary diseases. In this work, the interaction between H. pullorum and murine macrophages was examined. Firstly, the impact of nitric oxide, which is an antimicrobial produced by mammalian macrophages, on H. pullorum 6350-92 viability and morphology was studied by colony-forming assays and light microscopy, respectively. Exposure to nitric oxide lowered H. pullorum viability, in a growth-phase-dependent manner, and decreased the mean cell size. However, the number of coccoid forms remained low, contrasting with what has been observed for other Helicobacter species. Confocal microscopy showed that H. pullorum is internalized by murine macrophages, triggering nitric oxide production that promotes phagocytosis and killing of the pathogen. Interaction between H. pullorum and macrophages stimulated secretion of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and MIP-2. These results show that H. pullorum is able to infect mammalian murine cells triggering an inflammatory response.

Study of biofilm formation in C57Bl/6J mice by clinical isolates of Helicobacter pylori.

Background/Aim:Despite the significant number of studies on H. pylori pathogenesis, not much data has been published concerning its ability to form biofilm in the host stomach. This study aims to evaluate the potential of clinical isolates of H. pylori to form biofilm in C57BL/6J mice model.Materials and Methods:Two strains of H. pylori were selected from a collection of clinical isolates; one (19B), an efficient biofilm producer and the other (4B), with weak biofilm-forming ability. Mice infected through gastric avages were examined after one and two weeks. Colonization was determined by CFU and urease activity; the anti-H. pylori IgA was measured by ELISA, and chronic infections were evaluated by histopathology. Bacterial communities within mucosal sections were studied by immunofluorescence and scanning electron microscopy (SEM).Results:Successful infection was obtained by both test strains. Strain 19B with higher ability to form biofilm in vitro also showed a higher colonization rate in the mice stomach one week after infection. Difference (P < 0.05) in IgA titers was observed between the infected mice and the controls as well as between 19B and 4B infected mice, two weeks after the last challenge. Immunofluorescence and SEM results showed tightly colonizing H. pylori in stomach mucosal sections and in squamous and glandular epithelium.Conclusion:H. pylori is able to form biofilm in the mouse stomach and induce IgA production, reflecting the same potential as in humans. Firm attachment of coccoid form bacteria to host cells suggests the importance of this state in biofilm formation by H. pylori. Occurrence of biofilm in squamous and glandular epithelium of the mouse stomach proposes that H. pylori can all parts of the upper gastrointestinal tract.

Morphological and Bactericidal Effects of Amikacin, Meropenem and Imipenem on Pseudomonas aeruginosa.

Background:Pseudomonas aeruginosa might be converted to coccoid bacteria under antibiotic stress. Bacterial conversion would increase resistance to antibiotics due to changes in cell wall crosslink or decreased metabolic activity. Morphology of P. aeruginosa under stress conditions (presence of antibiotics) can be changed to elongated bacilli, U shape and finally coccoid bacteria. Results of several researches showed that coccoid bacteria are one of the most important aspects of drug resistance. It would be the major reason for treatment failure.Objectives:The aim of this study was to determine in vitro morphological and bactericidal effects of amikacin, meropenem and imipenem on P. aeruginosa isolated from clinical specimens.Materials and Methods:Eight P. aeruginosa isolates obtained from clinical samples of burned patients and standard strain ATCC 27853 were used in this study. Isolates were identified by biochemical tests and confirmed by PCR method using ITS specific primer. Minimum inhibitory concentrations (MICs) of three antibiotics were determined by E-test method. Bacteria were exposed to antibiotics at different concentrations. Bacterial morphology in different days was examined by specific microscope and viability of isolates was examined by flow cytometryResults:All used antibiotics at sub MIC concentration had capability to induce coccoid bacteria. The highest rate of induced coccoid bacteria was 98.2% after 8 days, with contribution of imipenem and meropenem at 2 μg/mL concentration. Amikacin at 4 μg/mL concentration induced lower rate of coccoid bacteria (55.05%). Amikacin had a strong bactericidal effect on coccoid bacteria at 8 μg/mL concentration. Imipenem and meropenem showed very weak bactericidal effect on coccoid bacteria.Conclusions:Induction of coccoid form of P. aeruginosa may be one of the important reasons for antibiotic treatment failure; therefore, prescribed dose of antibiotics should be carefully managed to prevent increasing antibiotic resistance and coccoid bacteria induction.

Answer to November 2015 Photo Quiz

Answer: Actinobaculum schaalii. The Gram stain of material from the abscess showed many Gram-positive cocci and Gram-negative coccobacilli. The organism that grew in aerobic culture initially presented as a pleomorphic Gram-positive organism with both coccoid and rod-shaped forms. After extended

Colonization of Lutzomyia verrucarum and Lutzomyia longipalpis Sand Flies (Diptera: Psychodidae) by Bartonella bacilliformis, the Etiologic Agent of Carrión's Disease.

Bartonella bacilliformis is a pathogenic bacterium transmitted to humans presumably by bites of phlebotomine sand flies, infection with which results in a bi-phasic syndrome termed Carrión’s disease. After constructing a low-passage GFP-labeled strain of B. bacilliformis, we artificially infected Lutzomyia verrucarum and L. longipalpis populations, and subsequently monitored colonization of sand flies by fluorescence microscopy. Initially, colonization of the two fly species was indistinguishable, with bacteria exhibiting a high degree of motility, yet still confined to the abdominal midgut. After 48h, B. bacilliformis transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species. Differences in colonization patterns became evident at 72h when B. bacilliformis was observed at relatively high density outside the peritrophic membrane in the lumen of the midgut in L. verrucarum, but colonization of L. longipalpis was limited to the blood meal within the intra-peritrophic space of the abdominal midgut, and the majority of bacteria were digested along with the blood meal by day 7. The viability of B. bacilliformis in L. longipalpis was assessed by artificially infecting, homogenizing, and plating for determination of colony-forming units in individual flies over a 13-d time course. Bacteria remained viable at relatively high density for approximately seven days, suggesting that L. longipalpis could potentially serve as a vector. The capacity of L. longipalpis to transmit viable B. bacilliformis from infected to uninfected meals was analyzed via interrupted feeds. No viable bacteria were retrieved from uninfected blood meals in these experiments. This study provides significant information toward understanding colonization of sand flies by B. bacilliformis and also demonstrates the utility of L. longipalpis as a user-friendly, live-vector model system for studying this severely neglected tropical disease.

Diagnostic methods for H. pylori infection: Choices, opportunities and pitfalls.

Diagnostic methods for H. pylori infection: Choices, opportunities and pitfalls.

Morphological Alteration and Survival of Burkholderia pseudomallei in Soil Microcosms.

The resilience of Burkholderia pseudomallei, the causative agent of melioidosis, was evaluated in control soil microcosms and in soil microcosms containing NaCl or FeSO4 at 30°C. Iron (Fe(II)) promoted the growth of B. pseudomallei during the 30-day observation, contrary to the presence of 1.5% and 3% NaCl. Scanning electron micrographs of B. pseudomallei in soil revealed their morphological alteration from rod to coccoid and the formation of microcolonies. The smallest B. pseudomallei cells were found in soil with 100 μM FeSO4 compared with in the control soil or soil with 0.6% NaCl (P < 0.05). The colony count on Ashdown's agar and bacterial viability assay using the LIVE/DEAD(®) BacLight(™) stain combined with flow cytometry showed that B. pseudomallei remained culturable and viable in the control soil microcosms for at least 120 days. In contrast, soil with 1.5% NaCl affected their culturability at day 90 and their viability at day 120. Our results suggested that a low salinity and iron may influence the survival of B. pseudomallei and its ability to change from a rod-like to coccoid form. The morphological changes of B. pseudomallei cells may be advantageous for their persistence in the environment and may increase the risk of their transmission to humans.

Rol de las formas cocoides de Helicobacter pylori en la infección y la recrudescencia

Helicobacter pylori is a spiral Gram-negative bacillus, which colonizes the human stomach and plays a key role in the pathogenesis of a number of gastroduodenal diseases. However, when expose to environmental stressed conditions, such as increased oxygen tension, extended incubation and exposure to antibiotics, Helicobacter pylori is able to entering the viable but nonculturable state, in which the bacterium modifies its morphology from a spiral to coccoid form, as a manifestation of cell adaptation to these adverse conditions. In gastric tissues, viable coccoid forms may remain latent for long time and retain virulence factors, so these forms possibly contribute to the treatment failures and recurrence of Helicobacter pylori infection and gastroduodenal diseases as well. In this review, we will discuss several aspects of cellular adaptation and survival of Helicobacter pylori, antibiotic susceptibility and virulence of coccoid forms and its involvement with recrudescence.

Methods for Detecting the Environmental Coccoid Form of Helicobacter pylori.

Helicobacter pylori is recognized as the most common pathogen to cause gastritis, peptic and duodenal ulcers, and gastric cancer. The organisms are found in two forms: (1) spiral-shaped bacillus and (2) coccoid. H. pylori coccoid form, generally found in the environment, is the transformed form of the normal spiral-shaped bacillus after exposed to water or adverse environmental conditions such as exposure to sub-inhibitory concentrations of antimicrobial agents. The putative infectious capability and the viability of H. pylori under environmental conditions are controversial. This disagreement is partially due to the fact of lack in detecting the coccoid form of H. pylori in the environment. Accurate and effective detection methods of H. pylori will lead to rapid treatment and disinfection, and less human health damages and reduction in health care costs. In this review, we provide a brief introduction to H. pylori environmental coccoid forms, their transmission, and detection methods. We further discuss the use of these detection methods including their accuracy and efficiency.

Stable isotope labeling by amino acids in cell culture based proteomics reveals differences in protein abundances between spiral and coccoid forms of the gastric pathogen Helicobacter pylori

Helicobacter pylori (H. pylori) is a ε-proteobacterium that colonizes the stomach of about half of the world's population. Persistent infections have been associated with several gastric diseases. Mainly rod- or spiral shaped but also coccoid H. pylori forms have been isolated from mucus layer biopsies of patients. It is still being debated whether the coccoid form can be transformed back into the spiral form or whether this morphology is a result of bacterial cell death or persistence.We established stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomics of H. pylori and applied it to investigate differences between the spiral and the coccoid morphology. We detected 72% and were able to relatively quantify 47% of the H. pylori proteome.Proteins involved in cell division and transcriptional and translational processes showed a lower abundance in coccoid cells. Additionally, proteins related to host colonization, including CagA, the arginase RocF, and the TNF-α inducing protein were down-regulated. The fact that outer membrane proteins were observed at higher abundances might represent a mechanism for immune evasion but also preserves adherence to host cells. The established protocol for relative protein quantification of H. pylori samples offers new possibilities for research on H. pylori. Biological significanceOur study shows that SILAC can be employed to study protein abundance changes in H. pylori. We have chosen to establish SILAC for H. pylori because it facilitates fractionation on both, protein and peptide level and thus enables deep proteome coverage. Furthermore, SILAC allows robust and highly accurate protein quantification. The manuscript includes a detailed description of the applied method, suggestions for further improvement as well as a practical application.The investigation of differences between the coccoid and infectious spiral morphology of H. pylori with SILAC revealed the regulation of proteins that are involved in host colonization, motility, cell division as well as transcriptional and translational processes. The data will help molecular biologist to focus on relevant pathways that were found to be regulated in response to morphological changes.Furthermore, the application of SILAC offers new possibilities to study the biology of H. pylori. It enables to monitor protein abundance changes in response to certain stimuli such as oxygen stress or antibiotics. Moreover, SILAC raises the possibility to study co-cultures of host cells and H. pylori on protein level. Additionally, pulsed SILAC experiments enable the quantification of protein turnover.

Cyanobacteria, algae and microfungi present in biofilm from Božana Cave (Serbia)

Phototrophic microorganisms (cyanobacteria and algae) and microfungi, were identified from biofilm on the walls of the entrance of Božana Cave in west Serbia. Temperature, relative humidity and light intensity were measured, and chlorophyll a content determined. Light intensity differed from the entrance inwards. However, Chl a content was not proportional to light intensity, instead it was positively correlated to biofilm weight. Biofilm samples from two sites were also observed using a scanning electron microscope. Coccoid forms of cyanobacteria were abundant at the sampling site with the lowest light intensity, while members of the order Nostocales were predominant at the sampling site with the highest light intensity measured. Cyanobacteria were the dominant group of phototrophs colonizing cave walls (29 taxa), with the order Chroococcales prevailing (21 taxa). The most frequently documented cyanobacteria were species from genera Gloeocapsa, Scytonema, Aphanocapsa and Chroococcus. Desmococcus olivaceus and Trentepohlia aurea were the only green algae documented on cave walls. Ascomycetes were common (e.g. Alternaria, Aspergillus, Cladosporium, Epicoccum, Penicillum and Trichoderma), while zygomycetes and oomycetes were less frequent. The different color of each biofilm sample was ascribed to the presence of various species of cyanobacteria and algae. biofilm; cave; chlorophil a; cave fungi; cave cyanobacteria