Campylobacter jejuni Virulence Factors Identified by Modulating Their Synthesis on Ribosomes With Altered rRNA Methylation

Campylobacter jejuni is a major cause of food poisoning worldwide, and remains the main infective agent in gastroenteritis and related intestinal disorders in Europe and the USA. As with all bacterial infections, the stages of adhesion to host tissue, survival in the host and eliciting disease all require the synthesis of proteinaceous virulence factors on the ribosomes of the pathogen. Here, we describe how C. jejuni virulence is attenuated by altering the methylation of its ribosomes to disrupt the composition of its proteome, and how this in turn provides a means of identifying factors that are essential for infection and pathogenesis. Specifically, inactivation of the C. jejuni Cj0588/TlyA methyltransferase prevents methylation of nucleotide C1920 in the 23S rRNA of its ribosomes and reduces the pathogen’s ability to form biofilms, to attach, invade and survive in host cells, and to provoke the innate immune response. Mass spectrometric analyses of C. jejuni TlyA-minus strains revealed an array of subtle changes in the proteome composition. These included reduced amounts of the cytolethal distending toxin (CdtC) and the MlaEFD proteins connected with outer membrane vesicle (OMV) production. Inactivation of the cdtC and mlaEFD genes confirmed the importance of their encoded proteins in establishing infection. Collectively, the data identify a subset of genes required for the onset of human campylobacteriosis, and serve as a proof of principle for use of this approach in detecting proteins involved in bacterial pathogenesis.

Positive Effect of Lactobacillus acidophilus EG004 on Cognitive Ability of Healthy Mice by Fecal Microbiome Analysis Using Full-Length 16S-23S rRNA Metagenome Sequencing

Recently, the concept of the “gut-brain axis” has risen and suggested that microbes in the GI tract affect the brain by modulating signal molecules. Although many pieces of research were reported in a short period, a signaling mechanism and the effects of a specific bacterial strain are still unclear.

Genomic Analysis and Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli in Peru

Campylobacter is the leading cause of human bacterial gastroenteritis worldwide and has a major impact on global public health. Whole Genome Sequencing (WGS) is a powerful tool applied in the study of foodborne pathogens. The objective of the present study was to apply WGS to determine the genetic diversity, virulence factors and determinants of antimicrobial resistance of the populations of C. jejuni and C. coli in Peru. A total of 129 Campylobacter strains (108 C. jejuni and 21 C. coli) were sequenced using Illumina Miseq platform. In silico MLST analysis identified a high genetic diversity among those strains with 30 sequence types (STs), several of them within 11 clonal complexes (CC) for C. jejuni, while the strains of C. coli belonged to a single CC with 8 different STs. Phylogeny analysis showed that Peruvian C. jejuni strains were divided into 2 clades with 5 populations, while C. coli formed a single clade with 4 populations. Furthermore, in silico analyses showed the presence of several genes associated with adherence, colonization and invasion among both species: cadF (83.7%), jlpA (81.4%), racR (100%), dnaJ (83.7%), pebA (83.7%), pldA (82.1%), porA (84.5%), ceuE (82.9%), ciaB (78.3%), iamB (86.8%), and flaC (100%). The majority (82.9%) of the Campylobacter strains carried the cdtABC operon which code for cytolethal distending toxin (CDT). Half of them (50.4%) carried genes associated with the presence of T6SS, while the frequency of genes associated with T4SS were relatively low (11.6%). Genetic markers associated with resistance to quinolones, tetracycline (tetO, tetW/N/W), beta-lactamases (blaoxa–61), macrolides (A2075G in 23S rRNA) were found in 94.5, 21.7, 66.7, 6.2, 69.8, and 18.6% of strains, respectively. The cmeABC multidrug efflux operon was present in 78.3% of strains. This study highlights the importance of using WGS in the surveillance of emerging pathogens associated with foodborne diseases, providing genomic information on genetic diversity, virulence mechanisms and determinants of antimicrobial resistance. The description of several Campylobacter genotypes having many virulence factors and resistance to quinolones and tetracyclines circulating in Peru provides important information which helps in the monitoring, control and prevention strategies of this emerging pathogen in our country.

Targeted editing and evolution of engineered ribosomes in vivo by filtered editing

Genome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into the E. coli genome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome’s translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.

Hemotropic mycoplasmas in bats from forest fragments, state of Paraná, southern Brazil

The order Chiroptera is the second largest group of mammals with bats being identified as reservoir of several viral zoonoses, although, little is known about their role in other groups of pathogens, including hemotropic Mycoplasma spp. To date, hemoplasma species have been found infecting several species of bats with high genetic diversity between 16S rRNA gene sequences. On this study, we aimed to identify the occurrence and characterize 16S and 23S rRNA genes of hemoplasma species in four bats species (Artibeus lituratus, Carollia perspicillata, Sturnira lilium and Sturnira tildae) from forest fragments in Paraná State, southern Brazil, using PCR-based assays. Spleen tissue samples were collected, DNA extracted and further screened by a pan‑hemoplasma PCR assay. All samples consistently amplified the mammal endogenous gapdh gene. One out of 15 (6.66%; 95% CI: 0.2-31%) bats tested positive for hemotropic Mycoplasma sp. by the PCR assay targeting the 16S rRNA gene. Sequencing of the 16S rRNA gene fragment from the hemoplasma-positive bat showed 99.14% identity with hemotropic Mycoplasma sp. detected in Sturnira parvidens from Belize. Sequencing of the 23S rRNA gene fragment from the hemoplasma-positive bat showed 86.17% identity with ‘Candidatus Mycoplasma haemosphiggurus’ detected in orange-spined hairy dwarf porcupines (Sphiggurus villosus) from Southern Brazil.

MinION™ Nanopore Sequencing of Skin Microbiome 16S and 16S-23S rRNA Gene Amplicons

Human skin microbiome dysbiosis can have clinical consequences. Characterizing taxonomic composition of bacterial communities associated with skin disorders is important for dermatological advancement in both diagnosis and novel treatments. This study aims to analyze and improve the accuracy of taxonomic classification of skin bacteria with MinION™ nanopore sequencing using a defined skin mock community and a skin microbiome sample. We compared the Oxford Nanopore Technologies recommended procedures and concluded that their protocols highly bias the relative abundance of certain skin microbiome genera, most notably a large overrepresentation of Staphylococcus and underrepresentation of Cutibacterium and Corynebacterium. We demonstrated that changes in the amplification protocols improved the accuracy of the taxonomic classification for these three main skin bacterial genera. This study shows that MinION™ nanopore could be an efficient technology for full-length 16S rRNA sequencing; however, the analytical advantage is strongly influenced by the methodologies. The suggested alternatives in the sample processing improved characterization of a complex skin microbiome community using MinION™ nanopore sequencing.

Water Age Effects on the Occurrence and Concentration of Legionella Species in the Distribution System, Premise Plumbing, and the Cooling Towers

In this study, droplet digital PCRTM (ddPCRTM) was used to characterize total Legionella spp. and five specific Legionella species from source (groundwater) to exposure sites (taps and cooling towers). A total of 42–10 L volume water samples were analyzed during this study: 12 from a reservoir (untreated groundwater and treated water storage tanks), 24 from two buildings (influents and taps), and six from cooling towers, all part of the same water system. The approximate water age (time in the system) for all sample locations are as follows: ~4.5, 3.4, 9.2, 20.8, and 23.2 h (h) for the groundwater to the reservoir influent, reservoir influent to the reservoir effluent, reservoir effluent to building Fa (building names are abbreviated to protect the privacy of site location), building ERC and the cooling towers, respectively. Results demonstrated that gene copies of Legionella spp. (23S rRNA) were significantly higher in the cooling towers and ERC building (p < 0.05) relative to the reservoir and building Fa (closest to reservoir). Legionella spp. (23S rRNA) were found in 100% (42/42) of water samples at concentrations ranging from 2.2 to 4.5 Log10 GC/100 mL. More specifically, L. pneumophila was found in 57% (24/42) of the water samples, followed by L. bozemanii 52% (22/42), L. longbeachae 36% (15/42), L. micdadei 23% (10/42) and L. anisa 21% (9/42) with geometric mean concentrations of 1.7, 1.7, 1.4, 1.6 and 1.7 Log10 GC/100 mL, respectively. Based on this study, it is hypothesized that water age in the distribution system and the premise-plumbing system as well as building management plays a major role in the increase of Legionella spp., (23S rRNA) and the diversity of pathogenic species found as seen in the influent, and at the taps in the ERC building—where the building water quality was most comparable to the industrial cooling towers. Other pathogenic Legionella species besides L.pneumophila are also likely amplifying in the system; thus, it is important to consider other disease relevant species in the whole water supply system—to subsequently control the growth of pathogenic Legionella in the built water environment.

Characterization of Lactic Bacteria Isolated from Raw Milk and Their Antibacterial Activity against Bacteria as the Cause of Clinical Bovine Mastitis

The objectives of this study were the selection of lactic acid bacteria (LAB) isolated from raw milk and studying their technological properties and antibacterial activities against bacteria as the cause of cattle mastitis. Biochemical and molecular identification using 16S–23S rRNA gene spacer analysis and 16S rRNA gene sequencing highlighted the presence of three species: Lactiplantibacillus plantarum, Lactococcus lactis, and Levilactobacillus brevis. The enzymatic characterization followed by the determination of technofunctional properties showed that LAB strains did not exhibit any hemolytic effect and were able to produce protease and lipase enzymes. Isolates showed very high antagonistic activity against Gram-positive and Gram-negative bacteria by producing H2O2, bacteriocin(s), and organic acid(s). APIZYM micromethod demonstrated that all selected strains are capable of producing valine arylamidase, cystine arylamidase, N-acetyl-β-glucosaminidase, and ᾳ-mannosidase. The antibiotic susceptibility assay showed that all selected strains were sensible to the majority of tested antibiotics. Based on these results, it can be concluded that the technological properties of the selected LAB allow considering their industrial use in order to formulate bioactive functional foods or drug(s).

Wolbachia Detection in Field-Collected Mosquitoes from Cameroon

Wolbachia spp., known to be maternally inherited intracellular bacteria, are widespread among arthropods, including mosquitoes. Our study assessed the presence and prevalence of Wolbachia infection in wild mosquitoes collected in Cameroon, using the combination of 23s rRNA Anaplasmatacea and 16s rRNA Wolbachia genes. Mosquitoes that were positive for Wolbachia were sequenced for subsequent phylogenetic analysis. Out of a total of 1740 individual mosquitoes belonging to 22 species and five genera screened, 33 mosquitoes (1.87%) belonging to eight species (namely, Aedes albopictus, A. contigus, Culex quinquefasciatus, C. perfuscus, C. wigglesworthi, C. duttoni, Anopheles paludis and Coquillettidia sp.) were found to be positive for Wolbachia infections. Wolbachia spp. were absent in A. gambiae and A. aegypti, the main vectors of malaria and dengue, respectively. Phylogenetic analysis of the 16S RNA sequences showed they belong mainly to two distinct subgroups (A and B). This study reports the presence of Wolbachia in about eight species of mosquitoes in Cameroon and suggests that future characterisation of the strains is needed.