Hydrogel Droplet Microarray for Genotyping Antimicrobial Resistance Determinants in Neisseria gonorrhoeae Isolates

A multiplex assay based on a low-density hydrogel microarray was developed to identify genomic substitutions in N. gonorrhoeae that determine resistance to the currently recommended treatment agents ceftriaxone and azithromycin and the previously used drugs penicillin, tetracycline, and ciprofloxacin. The microarray identifies 74 drug resistance determinants in the N. gonorrhoeae penA, ponA, porB, gyrA, parC, rpsJ, mtrR, blaTEM, tetM, and 23S rRNA genes. The hydrogel elements were formed by automated dispensing of nanoliter-volume droplets followed by UV-induced copolymerization of NH2-containing oligonucleotides with gel-forming monomers. Polybutylene terephthalate plates without special modifications were used as microarray substrates. Sequences and concentrations of immobilized oligonucleotides, gel composition, and hybridization conditions were carefully selected, and the median discrimination ratio ranged from 2.8 to 29.4, allowing unambiguous identification of single-nucleotide substitutions. The mutation identification results in a control sample of 180 N. gonorrhoeae isolates were completely consistent with the Sanger sequencing results. In total, 648 clinical N. gonorrhoeae isolates obtained in Russia during the last 5 years were analyzed and genotyped using these microarrays. The results allowed us to draw conclusions about the present situation with antimicrobial susceptibility of N. gonorrhoeae in Russia and demonstrated the possibility of using hydrogel microarrays to control the spread of antibiotic resistance.

BIOCOM-PIPE: a new user-friendly metabarcoding pipeline for the characterization of microbial diversity from 16S, 18S and 23S rRNA gene amplicons

Background The ability to compare samples or studies easily using metabarcoding so as to better interpret microbial ecology results is an upcoming challenge. A growing number of metabarcoding pipelines are available, each with its own benefits and limitations. However, very few have been developed to offer the opportunity to characterize various microbial communities (e.g., archaea, bacteria, fungi, photosynthetic microeukaryotes) with the same tool. Results BIOCOM-PIPE is a flexible and independent suite of tools for processing data from high-throughput sequencing technologies, Roche 454 and Illumina platforms, and focused on the diversity of archaeal, bacterial, fungal, and photosynthetic microeukaryote amplicons. Various original methods were implemented in BIOCOM-PIPE to (1) remove chimeras based on read abundance, (2) align sequences with structure-based alignments of RNA homologs using covariance models, and (3) a post-clustering tool (ReClustOR) to improve OTUs consistency based on a reference OTU database. The comparison with two other pipelines (FROGS and mothur) and Amplicon Sequence Variant definition highlighted that BIOCOM-PIPE was better at discriminating land use groups. Conclusions The BIOCOM-PIPE pipeline makes it possible to analyze 16S, 18S and 23S rRNA genes in the same packaged tool. The new post-clustering approach defines a biological database from previously analyzed samples and performs post-clustering of reads with this reference database by using open-reference clustering. This makes it easier to compare projects from various sequencing runs, and increased the congruence among results. For all users, the pipeline was developed to allow for adding or modifying the components, the databases and the bioinformatics tools easily, giving high modularity for each analysis.

Tracking Closely Related Enteric Bacteria at High Resolution in Fecal Samples of Premature Infants Using a Novel rRNA Amplicon

Identifying and tracking microbial strains as microbiomes evolve is a major challenge in the field of microbiome research. Longitudinal microbiome samples of co-admitted twins from two different neonatal intensive care units (NICUs) were analyzed using a ~2,500 base amplicon that spans the 16S and 23S rRNA genes, and mapped to a new 16S-23S rRNA database. Amplicon Sequence Variants inferred using DADA2 provided sufficient resolution for differentiation of rRNA variants from closely related, but not previously sequenced Klebsiella, E. coli, and Enterobacter, among the first bacteria colonizing the gut of these infants after admission to the NICU. Distinct ASV groups (fingerprints) were followed between co-admitted twins over time, demonstrating the potential to track the source and spread of both commensals and pathogens. The high-resolution taxonomy obtained from long amplicon sequencing enable tracking of strains temporally and spatially as microbiomes are established in infants in the hospital environment.

Genotypic and Phenotypic Resistance to Clarithromycin in Helicobacter pylori Strains

Background: The increasing prevalence of antimicrobial resistance, together with the lack of novel treatment options, negatively affects successful eradication of Helicobacter pylori. The aim of this study was to investigate genetic mutations in the 23S rRNA genes, which is associated with clarithromycin resistance, and to determine the clinical impact of genotype on phenotypic antimicrobial resistance. Methods: A total of 46 H. pylori strains were obtained from 13 patients, before and after unsuccessful eradication with clarithromycin-based triple therapy. The phenotypic resistance of each H. pylori strain was determined by minimum inhibitory concentration against clarithromycin using the serial two-fold agar dilution method. The genomic sequences of 23S rRNA genes were identified through next-generation sequencing, and nucleotide variants were determined based on comparison with genome sequences of the reference strain H. pylori 26695. Results: Clarithromycin resistance was found in 9 of 13 subjects before treatment and all subjects after unsuccessful eradication. Whole-genome sequencing of the 23S rRNA genes detected 42 mutations on 40 nonidentical loci, including 2147A>G (formerly 2143A>G) and 2146A>G (formerly 2142A>G). All strains with clarithromycin-resistant phenotype had either 2147A>G or 2146A>G mutation. When comparing genotype and phenotype for clarithromycin resistance, there was a significant association between 2147A>G mutation and clarithromycin-resistant phenotype. Conclusions: All clarithromycin-resistant strains had either 2146A>G or 2147A>G mutation, suggesting that tests targeting these two mutations may be enough for the prediction of clarithromycin resistance in this population.

Molecular detection of tick-borne pathogens in cattle from Saudi Arabia

Background: Babesiosis and anaplasmosis are tick-borne diseases that affect the health of cattle and may also be responsible for major economic losses. This study aimed to detect the presence of the causative agents of babesiosis and anaplasmosis among cattle in different provinces in Saudi Arabia and to characterise their species genetically. Methods: A total of 362 blood samples were taken from cattle in four regions (Riyadh, Al-Kharj, Al-Hasa and Al-Qassim) of Saudi Arabia and were molecularly screened by polymerase chain reaction (PCR) of partial 18S rRNA and 23S rRNA genes to detect Babesia and Anaplasma species. Results: The overall prevalence of Babesia and Anaplasma in cattle was 2.49% and 5.80%, respectively. Theileria annulata (T. annulata) and Theileria ovis (T. ovis) were found in seven (1.93%) and two (0.55%) of the 362 samples, respectively. Anaplasma ovis (A. ovis) was detected in 21 (5.80%) of 362 samples. Likewise, four of the cattle were found to be coinfected with more than one pathogen (1.10%). All cattle samples tested negative for other species of Babesia, Theileria and Anaplasma. Conclusions: The presence of T. ovis and A. ovis has been reported in Saudi Arabia. Here, the presence of two novel genotypes of T. annulata is also reported in Saudi Arabian cattle. Further molecular surveys on larger numbers of samples from the entire country are needed to correctly address the prevalence and geographical distribution of tick-borne disease.

Molecular detection of tick-borne pathogens in cattle from Saudi Arabia

Background and aim: Babesiosis and anaplasmosis are tick-borne diseases that affect the health of cattle and may be also responsible for remarkable economic losses. This study aimed to detect the presence of the causative agents of babesiosis and anaplasmosis among cattle in different provinces in Saudi Arabia and to characterise their species genetically. Methodology: A total of 362 blood samples were taken from cattle in four regions (Riyadh, Al-Kharj, Al-Hasa and Al-Qassim) of Saudi Arabia and were molecularly screened by polymerase chain reaction (PCR) of partial 18S rRNA and 23S rRNA genes to detect Babesia and Anaplasma species. Results The overall prevalence of both Babesia and Anaplasma in cattle was 2.49% and 5.80%, respectively. Theileria annulata (T. annulata) and Theileria ovis (T. ovis) were found in seven (1.93%) and two (0.55%) of the 362 samples, respectively. Anaplasma ovis (A. ovis) was detected in 21 (5.80%) of 362 samples. Likewise, four of the cattle were found to be co-infected with more than one pathogen (1.10%). All cattle samples tested negative for other species of Babesia, Theileria and Anaplasma. Conclusion The presence of T. ovis and A. ovis has been herein reported in Saudi Arabia. The presence of two novel genotypes of T. annulata is also reported in Saudi Arabian cattle. Further molecular surveys on larger numbers of samples from the entire country are needed in order to address correctly the prevalence and geographical distribution of the tick-borne disease.

Unlinked rRNA genes are widespread among Bacteria and Archaea

Ribosomes are essential to cellular life and the genes for their RNA components are the most conserved and transcribed genes in Bacteria and Archaea. These ribosomal rRNA genes are typically organized into a single operon, an arrangement that is thought to facilitate gene regulation. In reality, some Bacteria and Archaea do not share this canonical rRNA arrangement-their 16S and 23S rRNA genes are not co-located, but are instead separated across the genome and referred to as “unlinked”. This rearrangement has previously been treated as a rare exception or a byproduct of genome degradation in obligate intracellular bacteria. Here, we leverage complete genome and long-read metagenomic data to show that unlinked 16S and 23S rRNA genes are much more common than previously thought. Unlinked rRNA genes occur in many phyla, most significantly within Deinococcus-Thermus, Chloroflexi, Planctomycetes, and Euryarchaeota, and occur in differential frequencies across natural environments. We found that up to 41% of the taxa in soil, including dominant taxa, had unlinked rRNA genes, in contrast to the human gut, where all sequenced rRNA genes were linked. The frequency of unlinked rRNA genes may reflect meaningful life history traits, as they tend to be associated with a mix of slow-growing free-living species and obligatory intracellular species. Unlinked rRNA genes are also associated with changes in RNA metabolism, notably the loss of RNaseIII. We propose that unlinked rRNA genes may confer selective advantages in some environments, though the specific nature of these advantages remains undetermined and worthy of further investigation.

Genotypes and antibiotic resistance of bovineCampylobacterand their contribution to human campylobacteriosis

SUMMARYCampylobacter jejuniandCampylobacter coliare the most important bacterial causes of human gastroenteritis. Chicken has been recognized as a major source for human infection, whereas cattle might also contribute to a lesser extent. However, there is a paucity of information available regardingCampylobacterin Swiss cattle and their role for human campylobacteriosis. To gain more information on genotypes and antibiotic resistance of bovineC. jejuniandC. coliand on their contribution to human disease, 97 cattle isolates were analysed. Multilocus sequence typing (MLST) andflaBtyping were applied and thegyrAand 23S rRNA genes were screened for point mutations responsible for quinolone and macrolide resistance, respectively. A total of 37 sequence types (STs) and 44flaBtypes were identified, including two sequence types and fiveflaBtypes not previously described. Most common sequence types were ST21 (21%), ST61 (12%) and ST48 (11%). Only one isolate was macrolide resistant while 31% (n= 30) were quinolone resistant. Source attribution indicated chicken as the main source of human infection with cattle being second. In conclusion, cattle should not be underestimated as a potential source of human campylobacteriosis.