Generation of a CRISPR database for Yersinia pseudotuberculosis complex and role of CRISPR-based immunity in conjugation.

The clustered regularly interspaced short palindromic repeat - CRISPR-associated genes (CRISPR-Cas) system is used by bacteria and archaea against invading conjugative plasmids or bacteriophages. Central to this immunity system are genomic CRISPR loci that contain fragments of invading DNA. These are maintained as spacers in the CRISPR loci between direct repeats and the spacer composition in any bacterium reflects its evolutionary history. We analysed the CRISPR locus sequences of 335 Yersinia pseudotuberculosis complex strains. Altogether 1902 different spacer sequences were identified and these were used to generate a database for the spacer sequences. Only ∼10% of the spacer sequences found matching sequences. In addition, surprisingly few spacers were shared by Yersinia pestis and Y. pseudotuberculosis strains. Interestingly, 32 different protospacers were present in the conjugative plasmid pYptb32953. The corresponding spacers were identified from 35 different Y. pseudotuberculosis strains indicating that these strains had encountered pYptb32953 earlier. In conjugation experiments, pYptb32953-specific spacers generally prevented conjugation with spacer-positive and spacer-free strains. However, some strains with one to four spacers were invaded by pYptb32953 and some spacer-free strains were fully resistant. Also some spacer-positive strains were intermediate resistant to conjugation. This suggests that one or more other defence systems are determining conjugation efficiency independent of the CRISPR-Cas system.

Metagenomic Evaluation of Bacteria from Voles.

Voles (Arvicolinae, Rodentia) are known carriers of zoonotic bacteria such as Bartonella spp. and Francisella tularensis. However, apart from F. tularensis, the bacterial microbiome of voles has not previously been determined in Finland and rarely elsewhere. Therefore, we studied liver samples from 61 voles using 16S ribosomal RNA gene PCR analysis, followed by Sanger sequencing. Twenty-three of these samples were also studied with tag-encoded pyrosequencing. The samples originated from 21 field voles (Microtus agrestis), 37 tundra voles (Microtus oeconomus), and 3 bank voles (Myodes glareolus). With the more conventional 16S rDNA PCR analysis, 90 (33%) of the recovered 269 sequence types could be identified to genus level, including Bartonella, Francisella, Mycoplasma, Anaplasma, and Acinetobacter in 31, 15, 9, 9, and 9 sequences, respectively. Seventy-five (28%) matched best with sequences of uncultured bacteria, of which 40/75 could be classified to the order Clostridiales and, more specifically, to families Lachnospiraceae and Ruminococcaceae. Pyrosequencing from 23 samples revealed comparable and similar results: clinically relevant bacterial families such as Mycoplasmataceae, Bartonellaceae, Anaplasmataceae, and Francisellaceae were recognized. These analyses revealed significant bacterial diversity in vole livers, consisting of distinct and constant sequence patterns reflecting bacteria found in the intestinal gut, but including some known zoonotic pathogens as well. The molecular bacterial sequence types determined with the two different techniques shared major similarities and verified remarkable congruency between the methods.

The role of microbes in the pathogenesis of acute rhinosinusitis in young adults.

OBJECTIVES/HYPOTHESIS: To provide information on the course of acute rhinosinusitis (ARS) with sequential nasal and paranasal microbiological data and their correlation with clinical outcomes. STUDY DESIGN: We conducted a prospective cohort study among 50 Finnish military recruits with clinically diagnosed ARS in spring 2012. METHODS: We collected symptom, nasal endoscopy, and cone-beam CT (CBCT) scores during the early (2-3 days from onset) and later phases (9-10 days). We took viral samples from the nasopharynx (multiplex respiratory virus polymerase chain reaction [PCR]), bacterial culture from the middle meatus during both phases, and both viral and bacterial samples from the maxillary sinus aspirate (respiratory virus PCR, bacterial culture, broad-range bacterial PCR) during the later phase. Cilia destruction and microbial biofilms were sought from a nasal mucosal biopsy sample. RESULTS: We found that 42 (84%) of the subjects had viral nucleic acid in the nasopharynx during ARS. During the early phase, 28 (56%) of the subjects had nontypeable H. influenzae (NTHi) in the middle meatus, which was associated with wider paranasal mucosal changes in CBCT scans and increased symptoms during the study period. After 9 to 10 days from the onset, NTHi was found in the maxillary sinus in eight subjects (40%, 8/20) and led to prolonged symptoms. Bacterial biofilm was ruled out in 39 (78%) cases, and cilia destruction did not correlate with microbiological or clinical outcomes. CONCLUSION: Nasal and paranasal H. influenzae coinfection during viral infection may modify the symptoms and the extent of sinonasal mucosal disease observed in CBCT scans already from the beginning of the ARS episode.

Detection of Francisella tularensis in voles in Finland.

Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis-infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals.

Bacterial DNA signatures in carotid atherosclerosis represent both commensals and pathogens of skin origin.

Infectious agents have been suggested to be involved in atherosclerosis. By using a novel subtraction broad-range PCR approach, we defined bacterial DNA signatures in surgically removed sterile carotid artery endarterectomy plaques of patients with carotid atherosclerosis. Eighty partial bacterial 16S rDNA nucleotide sequences from eight patients were studied. Furthermore, 34 clones representing 21 bacterial sequence-types from the reagents used for DNA extraction and PCR amplification were determined. After subtraction of these potential methodological contaminants, 23 bacterial sequence-types were considered as clinically relevant findings. The most prominent phylum, Actinobacteria, accounted for 74% of these relevant sequences. Furthermore, according to the Human Microbiome project database, interestingly, nearly all (94%) of the sequences were associated with the human skin microbiome.

Detection of influenza A viruses with a portable real-time PCR instrument.

Timely identification of respiratory pathogens is essential for appropriate patient care and cohorting. In order to do rapid identification-technology near the patient we utilized the field-deployable RAZOR EX-thermocycler with a reverse transcription real-time PCR assay that detects all subtypes of influenza A virus. In addition, we developed a RT PCR assay for specific detection of influenza A(H1N1)pdm09 virus. These assays amplified segments of the matrix (M)- and the hemagglutinin (HA)-gene, respectively. Detection limits of the M-gene and the influenza A(H1N1)pdm09-specific HA-gene assays were 0.15 PFU and 8.8 PFU per reaction, respectively. With 18 influenza A viruses of different subtypes and influenza B, C, and 7 other respiratory viruses the RAZOR EX and standard real-time PCR assay results were in total agreement. From 104 clinical samples identical results were obtained by both PCR methods. Additional 21 clinical samples were tested under field conditions with the RAZOR EX instrument. Results were achieved in 90 min, including 45 min for sample preparation and they were in complete agreement with those obtained by standard real-time PCR under laboratory conditions. These methods enable highly sensitive and rapid on-site diagnostics to reliably identify patients infected with influenza A, including the influenza A(H1N1)pdm09-virus.

A multiplatform real-time polymerase chain reaction detection assay for Vibrio cholerae.

We report a multiplatform real-time polymerase chain reaction methodology based on genes encoding for the regulatory toxR activator and enterotoxin A protein to determine enterotoxigenic Vibrio cholerae types from other vibrios. This assay, which was successfully validated on a collection of 87 bacterial strains, including 63 representatives of V. cholerae and 8 noncholera vibrios provides a rapid tool for detection and identification of cholera.