Background: The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. Results: Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. Conclusion: In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.

Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient’s sequence clustered with sequences from Germany while the tourist’s sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients’ sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.

Background: Endemic presence of Klebsiella pneumoniae resistant to carbapenem in Italy has been due principally to the clonal expansion of CC258 isolates; however, recent studies suggest an ongoing epidemiological change in this geographical area. Methods: 50 K. pneumoniae strains, 25 carbapenem-resistant (CR-Kp) and 25 susceptible (CS-Kp), collected from march 2014 to march 2016 at the Laboratory of Bacteriology of the Paolo Giaccone Polyclinic University hospital of Palermo, Italy, were characterized for antibiotic susceptibility and fully sequenced by next generation sequencing (NGS) for the in silico analysis of resistome, virulome, multi-locus sequence typing (MLST) and core single nucleotide polymorphism (SNP) genotypes Results: MLST in silico analysis of CR-Kp showed that 52% of isolates belonged to CC258, followed by ST395 (12%), ST307 (12%), ST392 (8%), ST348 (8%), ST405 (4%) and ST101 (4%). In the CS-Kp group, the most represented isolate was ST405 (20%), followed by ST392 and ST15 (12%), ST395, ST307 and ST1727 (8%). The in silico β-lactamase analysis of the CR-Kp group showed that the most detected gene was blaSHV (100%), followed by blaTEM (92%), blaKPC (88%), blaOXA (88%) and blaCTX-M (32%). The virulome analysis detected mrk operon in all studied isolates, and wzi-2 was found in three CR-Kp isolates (12%). Furthermore, the distribution of virulence genes encoding for the yersiniabactin system, its receptor fyuA and the aerobactin system did not show significant distribution differences between CR-Kp and CS-Kp, whereas the Klebsiella ferrous iron uptake system (kfuA, kfuB and kfuC genes), the two-component system kvgAS and the microcin E495 were significantly (p < 0.05) prevalent in the CS-Kp group compared to the CR-Kp group. Core SNP genotyping, correlating with the MLST data, allowed greater strain tracking and discrimination than MLST analysis. Conclusions: Our data support the idea that an epidemiological change is ongoing in the Palermo area (Sicily, Italy). In addition, our analysis revealed the co-existence of antibiotic resistance and virulence factors in CR-Kp isolates; this characteristic should be considered for future genomic surveillance studies.

The 60 kDa molecular chaperones (chaperonins) are high molecular weight protein complexes having a characteristic double‐ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar, but distantly related families, one comprising the bacterial and organellar chaperonins and another (the so‐called CCT‐TRiC family) including the chaperonins of the archaea and the eukaryotes. Although some evidence exists that the archaeal chaperonins are implicated in protein folding, much remains to be learned about their precise cellular function. In this work, we report that the chaperonin of the thermophilic archaeon Sulfolobus solfataricus is an RNA‐binding protein that interacts specifically in vivo with the 16S rRNA and participates in the maturation of its 5′ extremity in vitro . We further show that the chaperonin binds RNA as the native heterooligomeric complex and that RNA binding and processing are inhibited by ATP. These results agree with previous reports indicating a role for the bacterial/organellar chaperonins in RNA protection or processing and suggest that all known chaperonin families share specific and evolutionarily ancient functions in RNA metabolism.

Background: Anthrax and plague are diseases caused by Bacillus anthracis and Yersinia pestis respectively. These bacteria are etiological agents for worldwide zoonotic diseases and are considered among the most feared potential bioterror agents. Strain differentiation is difficult for these microorganisms because of their high intraspecies genome homogeneity. Moreover, fast strain identification and comparison with known genotypes may be crucial for naturally occurring outbreaks versus bioterrorist events discrimination. Results: Thirty-nine B. anthracis and ten Y. pestis strains, representative of the species genetic diversity, were genotyped by Agilent 2100 Bioanalyzer using previously described Multiple Locus VNTR Analysis assays (MLVA). Results were compared to previous data obtained by standard genotyping system (capillary electrophoresis on automatic sequencer) and, when necessary, direct amplicon sequencing. A reference comparison table containing actual fragment sizes, sequencer sizes and Agilent sizes was produced. Conclusion: In this report an automated DNA electrophoresis apparatus which provides a cheaper alternative compared to capillary electrophoresis approaches was applied for genotyping of B. anthracis and Y. pesti s. This equipment, uses pre-cast gels and provides easy transportation, low maintenance and overall general logistic requirements and costs, is easy to set up and provides rapid analysis. This platform is a candidate for on-site MLVA genotyping of biothreat agents as well as other bacterial pathogens. It is an alternative to the more expensive and demanding capillary electrophoresis methods, and to the less expensive but more time-consuming classical gel electrophoresis approach.