Molecular evidence for a bacterium of the family Midichloriaceae (order Rickettsiales) in skin and organs of the rainbow trout Oncorhynchus mykiss affected by red mark syndrome

Red mark syndrome (RMS) is a chronic skin disease of unknown aetiology affecting farmed rainbow trout Oncorhynchus mykiss in Europe that causes single or multiple bright red skin lesions. Histological analysis showed acute inflammation in the area of the skin suggesting a bacterial infection. No aetiological agent has been unequivocally identified, although the involvement of a single transmissible agent has been suggested. The 16S rDNA of a bacterium belonging to the family Midichloriaceae (Rickettsiales) was found in association with RMS skin lesions.In this work, we present a novel specific method for absolute quantification of the midichloriacea associated with RMS in O. mykiss, based on a quantitative PCR approach. The qPCR method was tested on healthy skin, on lesions when present and on organ samples (heart, liver, spleen, intestine, kidney) from ten fish. Our work shows, for the first time, that the midichloriacea is present not only in skin lesions but also in organs of affected fish. Further studies are needed to prove whether this bacterium is actually involved in the pathology

Molecular screening for Midichloria bacteria in hard and soft ticks (Acari: Ixodida).

Ticks can harbor complex and highly variable microbial communities. Among these microorganisms,there are important pathogens of humans and animals that can be transmitted through the blood meal.Less is known about the other members of the microbial community of Ixodida, those that do not causeovert diseases. Among these, Midichloria mitochondrii, symbiont of the tick Ixodes ricinus, is the firstdescribed member of the family Midichloriaceae, order Rickettsiales. This bacterium is present in 100%females and is vertically transmitted (Sassera, 2008). The possibility of horizontal transmission issuggested by serological and molecular analyses showing positivity of mammalian blood and sera to M.mitochondrii (Mariconti, 2012; Bazzocchi, 2013). However, its role is still unknown. Recent reports areexpanding the view of this family, now including bacteria of great biological and medical interest,indicating a widespread distribution with an increasing range of hosts, with ticks being stronglyrepresented (Epis, 2008).Here we present a molecular screening of 17 tick species (for a total of 92 individuals), detecting andquantifying bacteria closely related to M. mitochondrii in seven of them, including the first report of amidichloriacea in a soft tick species, Ornithodoros maritimus. Based on sequence identity andphylogenetic analysis we propose that these bacteria could constitute the genus Midichloria. Theperformed screening highlights different prevalence levels in different tick species including one, Ixodesaulacodi, where the bacteria is present in all examined individuals, like in I. ricinus. This result promptsus to hypothesize different roles of Midichloria bacteria in different tick species

COL R Acinetobacter baumannii sRNA Signatures : Computational Comparative Identification and Biological Targets

Multidrug-Resistant (MDR) and Extensively Drug Resistant (XDR) Acinetobacter baumannii (Ab) represent a serious cause of healthcare-associated infections worldwide. Currently, the available treatment options are very restricted and colistin-based therapies are last-line treatments of these infections, even though colistin resistant (COL R) Ab have rarely been isolated yet. In bacteria, small non-coding RNAs (sRNAs) have been implicated in regulatory pathways of different biological functions, however, no knowledge exists about the sRNA role on the biological adaptation in COL R Ab. Our study investigated two Italian XDR isogenic colistin-susceptible/resistant (COL S/R) Ab strain-pairs to discover new sRNA signatures. Comparative sRNA transcriptome (sRNAome) analyses were carried out by Illumina RNA-seq using both a Tru-Seq and a Short Insert library, whilst Ab ATCC 17978 and ACICU Reference Genome assembly, mapping, annotation and statistically significant differential expression (q -value ≤ 0.01) of the raw reads were performed by the Rockhopper tool. A computational filtering, sorting only similarly statistically significant differentially expressed (DE) sRNAs mapping on the same gene in both COL R Ab isolates was conducted. COL R vs. COL S sRNAome, analyzed integrating the DE sRNAs obtained from the two different libraries, revealed some statistically significant DE sRNAs in COL R Ab. In detail, we found: (i) two different under-expressed cis -acting sRNAs (Ab sRNA and Ab sRNA) mapping in antisense orientation the 16S rRNA gene A1S_r01, (ii) one under-expressed cis -acting sRNA (Ab sRNA) targeting the A1S_2505 gene (hypothetical protein), (iii) one under-expressed microRNA-size small RNA fragment (Ab sRNA) and its pre-micro Ab sRNA targeting the A1S_0501 gene (hypothetical protein), (iv) as well as an over-expressed microRNA-size small RNA fragment (Ab sRNA) and its pre-micro Ab sRNA targeting the A1S_3097 gene (signal peptide). Custom TaqMan ® probe-based real-time qPCRs validated the expression pattern of the selected sRNA candidates shown by RNA-seq. Furthermore, analysis on sRNA ΔA1S_r01, ΔA1S_2505 as well as the over-expressed A1S_3097 mutants revealed no effects on colistin resistance. Our study, for the first time, found the sRNAome signatures of clinical COL R Ab with a computational prediction of their targets related to protein synthesis, host-microbe interaction and other different biological functions, including biofilm production, cell-cycle control, virulence, and antibiotic-resistance

Colistin Resistance Onset Strategies and Genomic Mosaicism in Clinical Acinetobacter baumannii Lineages

Abstract: The treatment of multidrug-resistant Gram-negative infections is based on colistin. As result, COL-resistance (COL-R) can develop and spread. In Acinetobacter baumannii, a crucial step is to understand COL-R onset and stability, still far to be elucidated. COL-R phenotypic stability, onset modalities, and phylogenomics were investigated in a clinical A. baumannii sample showing a COL resistant (COLR) phenotype at first isolation. COL-R was confirmed by Minimum-InhibitoryConcentrations as well as investigated by Resistance-Induction assays and Population-AnalysisProfiles (PAPs) to determine: (i) stability; (ii) inducibility; (iii) heteroresistance. Genomics was performed by Mi-Seq Whole-Genome-Sequencing, Phylogenesis, and Genomic Epidemiology by bioinformatics. COLR A. baumannii were subdivided as follows: (i) 3 A. baumannii with stable and high COL MICs defining the “homogeneous-resistant” onset phenotype; (ii) 6 A. baumannii with variable and lower COL MICs displaying a “COL-inducible” onset phenotype responsible for adaptive-resistance or a “subpopulation” onset phenotype responsible for COL-heteroresistance. COL-R stability and onset strategies were not uniquely linked to the amount of LPS and cell envelope charge. Phylogenomics categorized 3 lineages clustering stable and/or unstable COL-R phenotypes with increasing genomic complexity. Likewise, different nsSNP profiling in genes already associated with COL-R marked the stable and/or unstable COL-R phenotypes. Our investigation finds out that A. baumannii can range through unstable or stable COLR phenotypes emerging via different “onset strategies” within phylogenetic lineages displaying increasing genomic mosaicism

Demodicosis in a captive African straw-coloured fruit bat (Eidolon helvum).

Demodicosis is most frequently observed in the domestic dog (Canis familiaris), but it has rarely been reported in bats (Chiroptera). The overpopulation of Demodex spp. that causes dermatological changes is generally associated with a compromised immune system. We describe the gross and histological features of generalized demodicosis in an adult female African straw-coloured fruit bat (Eidolon helvum) drawn from a captive research colony. The histology of the lesions revealed comedones and follicular infundubular cysts harbouring numerous Demodex spp. mites, eliciting a minimal inflammatory response in the adjacent dermis. The histological examination of a full set of tissues did not reveal clear evidence of immunosuppression, although a clinical history of recent abortion and possible stressors due to captivity could be considered risk factors for the demodicosis. Attempts to determine the Demodex species using PCR on DNA extracted from the formalin fixed paraffin embedded tissue failed. This is the first clinical and histological description of demodicosis in Eidolon helvum

A Novel High Discriminatory Protocol for the Detection of Borrelia afzelii, Borrelia burgdorferi Sensu Stricto and Borrelia garinii in Ticks

Bacteria of the Borrelia burgdorferi sensu lato complex are the causative agents of Lyme borreliosis (LB). Even if the conventional diagnosis of LB does not rely on the species itself, an accurate species identification within the complex will provide a deepened epidemiological scenario, a better diagnosis leading to a more targeted therapeutic approach, as well as promote the general public's awareness. A comparative genomics approach based on the 210 Borrelia spp. genomes available in 2019 were used to set up three species-specific PCR protocols, able to detect and provide species typing of Borrelia afzelii, Borrelia burgdorferi sensu stricto (s.s.) and Borrelia garinii, the three most common and important human pathogenic Lyme Borrelia species in Europe. The species-specificity of these protocols was confirmed on previously identified B. afzelii, B. burgdorferi s.s. and B. garinii specimens detected in Ixodes ricinus samples. In addition, the protocols were validated on 120 DNA samples from ticks collected in Sweden, showing 88% accuracy, 100% precision, 72% sensitivity and 100% specificity. The proposed approach represents an innovative tool in epidemiological studies focused on B. burgdorferi s.l. occurrence in ticks, and future studies could suggest its helpfulness in routine diagnostic tests for health care

Molecular and Immunological Characterization of Staphylococcus aureus in Pediatric Atopic Dermatitis: Implications for Prophylaxis and Clinical Management

S. aureus represents a critical cofactor in atopic dermatitis (AD). In this paper, the prevalence of S. aureus infection/colonization was evaluated in 117 children as well as in their cohabitants, in order to assess the value of S. aureus characterization in predicting disease onset and severity and in providing indications for prophylaxis. Results showed that children with AD as well as their cohabitants had a significantly greater incidence of S. aureus infection/colonization as compared to controls. The genetic characterization showed a virtual identity of the bacteria strains collected at different sites of the patients with those found in the cohabitants, suggesting both a direct transmission between the nasal reservoir and the lesions in the same atopic subject and a risk for reinfection within family cohabitants. These data stress the need of preliminary laboratory assessment and posttherapy control in both AD patients and their close contacts for effective S. aureus eradication

Molecular screening for Midichloria in hard and soft ticks reveals variable prevalence levels and bacterial loads in different tick species

Candidatus Midichloria mitochondrii, symbiont of the sheep tick Ixodes ricinus, was the first described member of the family Candidatus Midichloriaceae, order Rickettsiales. Recent reports are expanding our view of this family, now including numerous bacteria of great biological and medical interest, indicating a widespread distribution with an increasing range of hosts, with ticks being strongly represented. Here we present a molecular screening of 17 tick species, detecting and quantifying bacteria of the family Midichloriaceae in seven of them, including the first report of a representative of this family in a soft tick species (Argasidae), Ornithodoros maritimus. Based on sequence identity and phylogenetic analysis we propose that all these bacterial symbionts of ticks could be members of the genus Midichloria. The performed screening highlights different prevalence levels and variable bacterial loads in different tick species including one, Ixodes aulacodi, where the bacterium is present in all examined individuals, like in I. ricinus. This result prompts us to hypothesize different roles of Midichloria bacteria in different tick species