Microorganisms, Volume 4, Issue 3 (September 2016) – 16 articles

Due to the increased number of immunocompromised patients, infections with the pathogen Candida albicans have significantly increased in recent years. C. albicans transition from yeast to germ tubes is one of the essential factors for virulence. In this study we noted that Lee’s medium, commonly used to induce filamentation, contained 500-fold more biotin than needed for growth and 40-fold more biotin than is typically added to growth media. Thus, we investigated the effects of excess biotin on growth rate and filamentation by C. albicans in different media. At 37 °C, excess biotin (4 µM) enhanced germ tube formation (GTF) ca. 10-fold in both Lee’s medium and a defined glucose-proline medium, and ca. 4-fold in 1% serum. Two biotin precursors, desthiobiotin and 7-keto-8-aminopelargonic acid (KAPA), also stimulated GTF. During these studies we also noted an inverse correlation between the number of times the inoculum had been washed and the concentration of serum needed to stimulate GTF. C. albicans cells that had been washed eight times achieved 80% GTF with only 0.1% sheep serum. The mechanism by which 1–4 µM biotin enhances GTF is still unknown except to note that equivalent levels of biotin are needed to create an internal supply of stored biotin and biotinylated histones. Biotin did not restore filamentation for any of the four known filamentation defective mutants tested. C. albicans is auxotrophic for biotin and this biotin auxotrophy was fulfilled by biotin, desthiobiotin, or KAPA. However, biotin auxotrophy is not temperature dependent or influenced by the presence of 5% CO₂. Biotin starvation upregulated the biotin biosynthetic genes BIO2, BIO3, and BIO4 by 11-, 1500-, and 150-fold, respectively, and BIO2p is predicted to be mitochondrion-localized. Based on our findings, we suggest that biotin has two roles in the physiology of C. albicans, one as an enzymatic cofactor and another as a morphological regulator. Finally, we found no evidence supporting prior claims that C. albicans only forms hyphae at very low biotin (0.1 nM) growth conditions. Full article

Biofilms formed by Staphylococcus aureus is a serious complication to the use of medical implants. A central part of the pathogenesis relies on S. aureus’ ability to adhere to host extracellular matrix proteins, which adsorb to medical implants and stimulate biofilm formation. Being coagulase positive, S. aureus furthermore induces formation of fibrin fibers from fibrinogen in the blood. Consequently, we hypothesized that fibrin is a key component of the extracellular matrix of S. aureus biofilms under in vivo conditions, and that the recalcitrance of biofilm infections can be overcome by combining antibiotic treatment with a fibrinolytic drug. We quantified S. aureus USA300 biofilms grown on peg-lids in brain heart infusion (BHI) broth with 0%–50% human plasma. Young (2 h) and mature (24 h) biofilms were then treated with streptokinase to determine if this lead to dispersal. Then, the minimal biofilm eradication concentration (MBEC) of 24 h old biofilms was measured for vancomycin and daptomycin alone or in combination with 10 µg/mL rifampicin in the presence or absence of streptokinase in the antibiotic treatment step. Finally, biofilms were visualized by confocal laser scanning microscopy. Addition of human plasma stimulated biofilm formation in BHI in a dose-dependent manner, and biofilms could be partially dispersed by streptokinase. The biofilms could be eradicated with physiologically relevant concentrations of streptokinase in combination with rifampicin and vancomycin or daptomycin, which are commonly used antibiotics for treatment of S. aureus infections. Fibronolytic drugs have been used to treat thromboembolic events for decades, and our findings suggest that their use against biofilm infections has the potential to improve the efficacy of antibiotics in treatment of S. aureus biofilm infections. Full article

Probiotics are live bacteria, generally administered in food, conferring beneficial effects to the host because they help to prevent or treat diseases, the majority of which are gastrointestinal. Numerous investigations have verified the beneficial effect of probiotic strains in biofilm form, including increased resistance to temperature, gastric pH and mechanical forces to that of their planktonic counterparts. In addition, the development of new encapsulation technologies, which have exploited the properties of biofilms in the creation of double coated capsules, has given origin to fourth generation probiotics. Up to now, reviews have focused on the detrimental effects of biofilms associated with pathogenic bacteria. Therefore, this work aims to amalgamate information describing the biofilms of Lactobacillus strains which are used as probiotics, particularly L. rhamnosus, L. plantarum, L. reuteri, and L. fermentum. Additionally, we have reviewed the development of probiotics using technology inspired by biofilms. Full article

Lactic acid bacteria (LAB) are Gram-positive bacteria that are natural inhabitants of the gastrointestinal (GI) tracts of mammals, including humans. Since Mechnikov first proposed that yogurt could prevent intestinal putrefaction and aging, the beneficial effects of LAB have been widely demonstrated. The region between the duodenum and the terminal of the ileum is the primary region colonized by LAB, particularly the Lactobacillus species, and this region is covered by a mucus layer composed mainly of mucin-type glycoproteins. The mucus layer plays a role in protecting the intestinal epithelial cells against damage, but is also considered to be critical for the adhesion of Lactobacillus in the GI tract. Consequently, the adhesion exhibited by lactobacilli on mucin has attracted attention as one of the critical factors contributing to the persistent beneficial effects of Lactobacillus in a constantly changing intestinal environment. Thus, understanding the interactions between Lactobacillus and mucin is crucial for elucidating the survival strategies of LAB in the GI tract. This review highlights the properties of the interactions between Lactobacillus and mucin, while concomitantly considering the structure of the GI tract from a histochemical perspective. Full article

Achromobacter species have attracted attention as emerging pathogens in cystic fibrosis. The clinical significance of Achromobacter infection is not yet fully elucidated; however, their intrinsic resistance to antimicrobials and ability to form biofilms renders them capable of establishing long-term chronic infections. Still, many aspects of Achromobacter biofilm formation remain uncharacterized. In this study, we characterized biofilm formation in clinical isolates of Achromobacter and investigated the effect of challenging the biofilm with antimicrobials and/or enzymes targeting the extracellular matrix. In vitro biofilm growth and subsequent visualization by confocal microscopy revealed distinctly different biofilm morphotypes: a surface-attached biofilm morphotype of small aggregates and an unattached biofilm morphotype of large suspended aggregates. Aggregates consistent with our in vitro findings were visualized in sputum samples from cystic fibrosis patients using an Achromobacter specific peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) probe, confirming the presence of Achromobacter biofilms in the CF lung. High antibiotic tolerance was associated with the biofilm phenotype, and biocidal antibiotic concentrations were up to 1000 fold higher than for planktonic cultures. Treatment with DNase or subtilisin partially dispersed the biofilm and reduced the tolerance to specific antimicrobials, paving the way for further research into using dispersal mechanisms to improve treatment strategies. Full article

Sequencing surveys of microbial communities in marine subsurface sediments have focused on organic-rich, continental margins; the database for organic-lean deep-sea sediments from mid-ocean regions is underdeveloped. The archaeal community in subsurface sediments of ODP Site 1225 in the eastern equatorial Pacific (3760 m water depth; 1.1 and 7.8 m sediment depth) was analyzed by PCR, cloning and sequencing, and by denaturant gradient gel electrophoresis (DGGE) of 16S rRNA genes. Three uncultured archaeal lineages with different depth distributions were found: Marine Group I (MG-I) within the Thaumarchaeota, its sister lineage Marine Benthic Group A (MBG-A), the phylum-level archaeal lineage Marine Benthic Group B (also known as Deep-Sea Archaeal Group or Lokiarchaeota), and the Deep-Sea Euryarchaeotal Group 3. The MG-I phylotypes included representatives of sediment clusters that are distinct from the pelagic members of this phylum. On the scale from fully oxidized, extremely organic carbon-depleted sediments (for example, those the South Pacific Gyre) to fully reduced, organic carbon-rich marine subsurface sediments (such as those of the Peru Margin), Ocean Drilling Program (ODP) Site 1225 falls into the non-extreme organic carbon-lean category, and harbors archaeal communities from both ends of the spectrum. Full article

Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Neisseria meningitidis is a closely related pathogen that shares many of the same genomic features and virulence factors, but causes the life threatening diseases meningococcal meningitis and septicaemia. The importance of non-coding RNAs in gene regulation has become increasingly evident having been demonstrated to be involved in regulons responsible for iron acquisition, antigenic variation, and virulence. Neisseria spp. contain an IS-like element, the Correia Repeat Enclosed Element, which has been predicted to be mobile within the genomes or to have been in the past. This repeat, present in over 100 copies in the genome, has the ability to alter gene expression and regulation in several ways. We reveal here that Correia Repeat Enclosed Elements tend to be near non-coding RNAs in the Neisseria spp., especially N. gonorrhoeae. These results suggest that Correia Repeat Enclosed Elements may have disrupted ancestral regulatory networks not just through their influence on regulatory proteins but also for non-coding RNAs. Full article

Reactive (inflammatory) arthritis has been known for many years to follow genital infection with the intracellular bacterial pathogen Chlamydia trachomatis in some individuals. Recent studies from several groups have demonstrated that a related bacterium, the respiratory pathogen Chlamydia pneumoniae, can elicit a similar arthritis. Studies of these organisms, and of a set of gastrointestinal pathogens also associated with engendering inflammatory arthritis, have been relatively extensive. However, reports focusing on coinfections with these and/or other organisms, and the effects of such coinfections on the host immune and other systems, have been rare. In this article, we review the extant data regarding infections by multiple pathogens in the joint as they relate to engendering arthritis, and we suggest a number of research areas that must be given a high priority if we are to understand, and therefore to treat in an effective manner, such arthritides. Full article

Horizontal gene transfer (HGT) is a driving force to the evolution of bacteria. The fast emergence of antimicrobial resistance reflects the ability of genetic adaptation of pathogens. Acinetobacter baumannii has emerged in the last few decades as an important opportunistic nosocomial pathogen, in part due to its high capacity of acquiring resistance to diverse antibiotic families, including to the so-called last line drugs such as carbapenems. The rampant selective pressure and genetic exchange of resistance genes hinder the effective treatment of resistant infections. A. baumannii uses all the resistance mechanisms to survive against carbapenems but production of carbapenemases are the major mechanism, which may act in synergy with others. A. baumannii appears to use all the mechanisms of gene dissemination. Beyond conjugation, the mostly reported recent studies point to natural transformation, transduction and outer membrane vesicles-mediated transfer as mechanisms that may play a role in carbapenemase determinants spread. Understanding the genetic mobilization of carbapenemase genes is paramount in preventing their dissemination. Here we review the carbapenemases found in A. baumannii and present an overview of the current knowledge of contributions of the various HGT mechanisms to the molecular epidemiology of carbapenem resistance in this relevant opportunistic pathogen. Full article

Ticks carry several human pathogenic microbes including Borreliae and Flavivirus causing tick-born encephalitis. Ticks can also carry DNA of Chlamydia-like organisms (CLOs). The purpose of this study was to investigate the occurrence of CLOs in ticks and skin biopsies taken from individuals with suspected tick bite. DNA from CLOs was detected by pan-Chlamydiales-PCR in 40% of adult ticks from southwestern Finland. The estimated minimal infection rate for nymphs and larvae (studied in pools) was 6% and 2%, respectively. For the first time, we show CLO DNA also in human skin as 68% of all skin biopsies studied contained CLO DNA as determined through pan-Chlamydiales-PCR. Sequence analyses based on the 16S rRNA gene fragment indicated that the sequences detected in ticks were heterogeneous, representing various CLO families; whereas the majority of the sequences from human skin remained “unclassified Chlamydiales” and might represent a new family-level lineage. CLO sequences detected in four skin biopsies were most closely related to “uncultured Chlamydial bacterium clones from Ixodes ricinus ticks” and two of them were very similar to CLO sequences from Finnish ticks. These results suggest that CLO DNA is present in human skin; ticks carry CLOs and could potentially transmit CLOs to humans. Full article

Researchers have demonstrated that lactic acid bacteria (LAB) with immunomodulatory capabilities (immunobiotics) exert their beneficial effects through several molecules, including cell wall, peptidoglycan, and exopolysaccharides (EPS), that are able to interact with specific host cell receptors. EPS from LAB show a wide heterogeneity in its composition, meaning that biological properties depend on the strain and. therefore, only a part of the mechanism of action has been elucidated for these molecules. In this review, we summarize the current knowledge of the health-promoting actions of EPS from LAB with special focus on their immunoregulatory actions. In addition, we describe our studies using porcine intestinal epithelial cells (PIE cells) as a model to evaluate the molecular interactions of EPS from two immunobiotic LAB strains and the host cells. Our studies showed that EPS from immunobiotic LAB have anti-inflammatory capacities in PIE cells since they are able to reduce the production of inflammatory cytokines in cells challenged with the Toll-like receptor (TLR)-4-agonist lipopolysaccharide. The effects of EPS were dependent on TLR2, TLR4, and negative regulators of TLR signaling. We also reported that the radioprotective 105 (RP105)/MD1 complex, a member of the TLR family, is partially involved in the immunoregulatory effects of the EPS from LAB. Our work described, for the first time, that LAB and their EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependent manner. A continuing challenge for the future is to reveal more effector-receptor relationships in immunobiotic-host interactions that contribute to the beneficial effects of these bacteria on mucosal immune homeostasis. A detailed molecular understanding should lead to a more rational use of immunobiotics in general, and their EPS in particular, as efficient prevention and therapies for specific immune-related disorders in humans and animals. Full article

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1–35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation. Full article

The intracellular life style of chlamydia and the ability to cause persistent infections with low-grade replication requires tests with high analytical sensitivity to directly detect C. trachomatis (CT) in medical samples. Nucleic acid amplification tests (NAATs) are the most sensitive assays with a specificity similar to cell culture and are considered the method of choice for CT detection. In addition, NAATs can be performed on various clinical specimens that do not depend on specific transport and storage conditions, since NAATs do not require infectious bacteria. In the case of lower genital tract infections, first void urine and vaginal swabs are the recommended specimens for testing males and females, respectively. Infections of anorectal, oropharyngeal and ocular epithelia should also be tested by NAAT analysis of corresponding mucosal swabs. In particular, anorectal infections of men who have sex with men (MSM) should include evaluation of lymphogranuloma venereum (LGV) by identification of genotypes L1, L2 or L3. Detection of CT antigens by enzyme immunoassay (EIAs) or rapid diagnostic tests (RDTs) are unsuitable due to insufficient sensitivity and specificity. Recent PCR-based RDTs, however, are non-inferior to standard NAATs, and might be used at the point-of-care. Serology finds application in the diagnostic work-up of suspected chronic CT infection but is inappropriate to diagnose acute infections. Full article

The Deepwater Horizon (DWH) oil spill led to rapid microbial community shifts in the Gulf of Mexico, including the formation of unprecedented quantities of marine oil snow (MOS) and of a massive subsurface oil plume. The major taxa that bloomed in sea surface oil slicks during the spill included Cycloclasticus, and to a lesser extent Halomonas, Alteromonas, and Pseudoalteromonas—organisms that grow and degrade oil hydrocarbons aerobically. Here, we show that sea surface oil slicks at DWH contained obligate and facultative anaerobic taxa, including members of the obligate anaerobic phylum Fusobacteria that are commonly found in marine sediment environments. Pyrosequencing analysis revealed that Fusobacteria were strongly selected for when sea surface oil slicks were allowed to develop anaerobically. These organisms have been found in oil-contaminated sediments in the Gulf of Mexico, in deep marine oil reservoirs, and other oil-contaminated sites, suggesting they have putative hydrocarbon-degrading qualities. The occurrence and strong selection for Fusobacteria in a lab-based incubation of a sea surface oil slick sample collected during the spill suggests that these organisms may have become enriched in anaerobic zones of suspended particulates, such as MOS. Whilst the formation and rapid sinking of MOS is recognised as an important mechanism by which a proportion of the Macondo oil had been transported to the sea floor, its role in potentially transporting microorganisms, including oil-degraders, from the upper reaches of the water column to the seafloor should be considered. The presence of Fusobacteria on the sea surface—a highly oxygenated environment—is intriguing, and may be explained by the vertical upsurge of oil that provided a carrier to transport these organisms from anaerobic/micro-aerophilic zones in the oil plume or seabed to the upper reaches of the water column. We also propose that the formation of rapidly-sinking MOS may have re-transported these, and other microbial taxa, to the sediment in the Gulf of Mexico. Full article

Antillogorgia elisabethae (synonymous with Pseudopterogorgia elisabethae) is a common branching octocoral in Caribbean reef ecosystems. A. elisabethae is a rich source of anti-inflammatory diterpenes, thus this octocoral has been the subject of numerous natural product investigations, yet relatively little is known regarding the composition, diversity and the geographic and temporal stability of its microbiome. To characterize the composition, diversity and stability of bacterial communities of Bahamian A. elisabethae populations, 17 A. elisabethae samples originating from five sites within The Bahamas were characterized by 16S rDNA pyrosequencing. A. elisabethae bacterial communities were less diverse and distinct from those of surrounding seawater samples. Analyses of α- and β-diversity revealed that A. elisabethae bacterial communities were highly variable between A. elisabethae samples from The Bahamas. This contrasts results obtained from a previous study of three specimens collected from Providencia Island, Colombia, which found A. elisabethae bacterial communities to be highly structured. Taxa belonging to the Rhodobacteriales, Rhizobiales, Flavobacteriales and Oceanospiralles were identified as potential members of the A. elisabethae core microbiome. Full article

Listeria monocytogenes is a foodborne pathogen able to persist in food industry and is responsible for a severe illness called listeriosis. The ability of L. monocytogenes to persist in environments is due to its capacity to form biofilms that are a sessile community of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS’s). In this review, we summarized recent efforts performed in order to better characterize the polymeric substances that compose the extracellular matrix (ECM) of L. monocytogenes biofilms. EPS extraction and analysis led to the identification of polysaccharides, proteins, extracellular DNA, and other molecules within the listerial ECM. All this knowledge will be useful for increasing food protection, suggesting effective strategies for the minimization of persistence of L. monocytogenes in food industry environments. Full article