Mucin induces CRISPR-Cas defense in an opportunistic pathogen.

Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we co-evolved the bacterial fish pathogen Flavobacterium columnare and its virulent phage V156 in presence and absence of a eukaryotic host signal (mucin) for sixteen weeks. The presence of mucin leads to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies obtain at least one new spacer. Additionally, we show that the presence of a competitor bacterium further increases CRISPR spacer acquisition in F. columnare. These results suggest that ecological factors are important in determining defense strategies against phages, and that the phage-bacterium interactions on mucosal surfaces may select for the diversification of bacterial immune systems.

Mucosal Environment Induces Phage Susceptibility in Streptococcus mutans.

Pathogenic bacteria are attracted toward mucosa, as it is their way of entry into the body. However, we know surprisingly little about the phage-bacterium interactions in the mucosal environment. Here, we explored the effect of the mucosal environment on growth characteristics and phage-bacterium interactions in Streptococcus mutans, a causative agent of dental caries. We found that although mucin supplementation increased bacterial growth and survival, it decreased S. mutans biofilm formation. More importantly, the presence of mucin had a significant effect on S. mutans phage susceptibility. In two experiments done in Brain Heart Infusion Broth, phage M102 replication was detected only with 0.2% mucin supplementation. In 0.1 × Tryptic Soy Broth, 0.5% mucin supplementation led to a 4-log increase in phage titers compared with the control. These results suggest that the mucosal environment can have a major role in the growth, phage sensitivity, and phage resistance of S. mutans, and underline the importance of understanding the effect of mucosal environment on phage-bacterium interactions.

Imaging Bacterial Colonies and Phage-Bacterium Interaction at Sub-Nanometer Resolution Using Helium-Ion Microscopy.

Imaging of microbial interactions has so far been based on well-established electron microscopy methods. This study presents a new way to study bacterial colonies and interactions between bacteria and their viruses, bacteriophages (phages), in situ on agar plates using helium ion microscopy (HIM). In biological imaging, HIM has advantages over traditional scanning electron microscopy with its sub-nanometer resolution, increased surface sensitivity, and the possibility to image nonconductive samples. Furthermore, by controlling the He beam dose or by using heavier Ne ions, the HIM instrument provides the possibility to mill out material in the samples, allowing for subsurface imaging and in situ sectioning. Here, the first HIM-images of bacterial colonies and phage-bacterium interactions are presented at different stages of the infection as they occur on an agar culture. The feasibility of neon and helium milling is also demonstrated to reveal the subsurface structures of bacterial colonies on agar substrate, and in some cases also structure inside individual bacteria after cross-sectioning. The study concludes that HIM offers great opportunities to advance the studies of microbial imaging, in particular in the area of interaction of viruses with cells.

Molecular evidence of Orthopoxvirus DNA in capybara (Hydrochoerus hydrochaeris) stool samples.

Vaccinia virus (VACV) is responsible for outbreaks in Brazil and has immense potential as an emerging virus. VACV can be found naturally circulating in India, Pakistan and South America, where it causes infections characterised by exanthematic lesions in buffaloes, cattle and humans. The transmission cycle of Brazilian VACV has still not been fully characterised; one of the most important gaps in knowledge being the role of wild animals. Capybaras, which are restricted to the Americas, are the world's largest rodents and have peculiar characteristics that make them possible candidates for being part of a natural VACV reservoir. Here, we developed a method for detecting orthopoxvirus DNA in capybara stool samples, and have described for the first time the detection of orthopoxvirus DNA in capybaras samples from three different regions in Brazil. These findings strongly suggest that capybaras might be involved in the natural transmission cycle of VACV and furthermore represent a public health problem, when associated with Brazilian bovine vaccinia outbreaks. This makes infected animals an important factor to be considered when predicting and managing Brazilian VACV outbreaks.

Complete genome sequences of Francisella noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190: a fish pathogen with genomic clonal behavior.

The genus Francisella is composed of Gram-negative, pleomorphic, strictly aerobic and non-motile bacteria, which are capable of infecting a variety of terrestrial and aquatic animals, among which Francisella noatunensis subsp. orientalis stands out as the causative agent of pyogranulomatous and granulomatous infections in fish. Accordingly, F. noatunensis subsp. orientalis is responsible for high mortality rates in freshwater fish, especially Nile Tilapia. In the current study, we present the genome sequences of F. noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190. The genomes include one circular chromosome of 1,859,720 bp, consisting of 32 % GC content, 1538 coded proteins and 363 pseudogenes for FNO12; one circular chromosome of 1,862,322 bp, consisting of 32 % GC content, 1537 coded proteins and 365 pseudogenes for FNO24; and one circular chromosome of 1,859,595 bp, consisting of 32 % GC content, 1539 coded proteins and 362 pseudogenes for FNO190. All genomes have similar genetic content, implicating a clonal-like behavior for this species.

High positivity of mimivirus in inanimate surfaces of a hospital respiratory-isolation facility, Brazil.

BACKGROUND: Mimiviruses have been considered putative emerging pneumonia agents. Pneumonia is a leading cause of death related to infection throughout the world, with approximately 40% of cases presenting unknown etiology. Therefore, identifying new causative agents of community and nosocomial pneumonia is of major public health concern. OBJECTIVE: We evaluated the distribution of these viruses in samples collected from different environments of one of the largest hospitals in Brazilian Southeast. STUDY DESIGN: We analyzed, by molecular and virological approaches, the distribution of mimivirus in 242 samples collected from inanimate surfaces in different hospital facilities. RESULTS: A significant positivity of mimivirus in respiratory-isolation-facilities was observed (p<0.001). CONCLUSION: Although the role of mimivirus as etiological agents of pneumonia is still under investigation, our results demonstrates interesting correlations that strengthens the need for control over the occurrence of these viruses in hospital facilities.

Differential upregulation of human 2'5'OAS genes on systemic sclerosis: Detection of increased basal levels of OASL and OAS2 genes through a qPCR based assay.

2'5'OAS are template-independent RNA polymerases with antiviral activity and important to homeostasis maintenance. Here we have developed quantitative PCR (qPCR) reactions for the detection of each individual 2'5'OAS human gene and used them to evaluate these gene levels in systemic sclerosis patients cells. The method was efficient for quantification of 2'5'OAS genes on human cells after interferon (IFN) treatment, and revealed that primary cells from patients with systemic sclerosis have increased basal levels of OASL and OAS2 genes. When treated, patients cells are able to induce all four 2'5'OAS genes. Our hypothesis is that abnormally circulating type I IFNs on the disease could be establishing a desensitized state on patients cells, making them refractory to subsequent IFN doses, and that OASL and OAS2 genes upregulation may be due to an IFN-independent stimulus. Further characterizing the biological activities of these genes, their induction pathways and their regulatory functions can lead to better understanding of systemic sclerosis molecular mechanisms and of their biological activities.