RESUMEN

BACKGROUND: Campylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide. METHODS: We systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids. RESULTS: Overall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3')-IIIa, aph(2'')-If, catA, lnu(C), blaOXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter. CONCLUSIONS: The results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety.

Asunto(s)

Infecciones por Campylobacter , Campylobacter , Humanos , Infecciones por Campylobacter/microbiología , Farmacorresistencia Bacteriana/genética , Antibacterianos/farmacología , Campylobacter/genética , Gentamicinas , Secuenciación Completa del Genoma , Pruebas de Sensibilidad Microbiana

RESUMEN

Penicillin-resistant Streptococcus pneumoniae strains are found at high rates in Romania and Iran. The mosaic structure of PBP2x was investigated in 9 strains from Iran and in 15 strains from Romania to understand their evolutionary history. Mutations potentially important for ß-lactam resistance were identified by comparison of the PBP2x sequences with the sequence of the related PBP2x of reference penicillin-sensitive S. mitis strains. Two main PBP2x mosaic gene families were recognized. Eight Iranian strains expressed PBP2x variants in group 1, which had a mosaic block highly related to PBP2x of the Spain23F-1 clone, which is widespread among international penicillin-resistant S. pneumoniae clones. A second unique PBP2x group was observed in Romanian strains; furthermore, three PBP2x single mosaic variants were found. Sequence blocks of penicillin-sensitive strain S. mitis 658 were common among PBP2x variants from strains from both countries. Each PBP2x group contained specific signature mutations within the transpeptidase domain, documenting the existence of distinct mutational pathways for the development of penicillin resistance.

Asunto(s)

Antibacterianos/farmacología , Mosaicismo , Resistencia a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/genética , Penicilinas/farmacología , Streptococcus pneumoniae/genética , Anciano , Secuencia de Aminoácidos , Niño , Preescolar , Células Clonales , Femenino , Expresión Génica , Humanos , Lactante , Irán , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Modelos Moleculares , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/metabolismo , Polimorfismo Genético , Rumanía , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Infecciones Estreptocócicas/tratamiento farmacológico , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/patología , Streptococcus mitis/efectos de los fármacos , Streptococcus mitis/genética , Streptococcus mitis/aislamiento & purificación , Streptococcus mitis/metabolismo , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/aislamiento & purificación , Streptococcus pneumoniae/metabolismo , Adulto Joven

RESUMEN

The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales), is a widespread, vertically transmitted endosymbiont of filarial nematodes and arthropods. In insects, Wolbachia modifies reproduction, and in mosquitoes, infection interferes with replication of arboviruses, bacteria and plasmodia. Development of Wolbachia as a tool to control pest insects will be facilitated by an understanding of molecular events that underlie genetic exchange between Wolbachia strains. Here, we used nucleotide sequence, transcriptional and proteomic analyses to evaluate expression levels and establish the mosaic nature of genes flanking the T4SS virB8-D4 operon from wStr, a supergroup B-strain from a planthopper (Hemiptera) that maintains a robust, persistent infection in an Aedes albopictus mosquito cell line. Based on protein abundance, ribA, which contains promoter elements at the 5'-end of the operon, is weakly expressed. The 3'-end of the operon encodes an intact wspB, which encodes an outer membrane protein and is co-transcribed with the vir genes. WspB and vir proteins are expressed at similar, above average abundance levels. In wStr, both ribA and wspB are mosaics of conserved sequence motifs from Wolbachia supergroup A- and B-strains, and wspB is nearly identical to its homolog from wCobU4-2, an A-strain from weevils (Coleoptera). We describe conserved repeated sequence elements that map within or near pseudogene lesions and transitions between A- and B-strain motifs. These studies contribute to ongoing efforts to explore interactions between Wolbachia and its host cell in an in vitro system.

Asunto(s)

Genes Bacterianos/genética , Wolbachia/genética , Animales , Secuencia de Bases , Operón/genética , Proteómica

RESUMEN

Intragenic recombination leading to mosaic gene formation is known to alter resistance profiles for particular genes and bacterial species. Few studies have examined to what extent aminoglycoside resistance genes undergo intragenic recombination. We screened the GenBank database for mosaic gene formation in homologs of the aph(3')-IIa (nptII) gene. APH(3')-IIa inactivates important aminoglycoside antibiotics. The gene is widely used as a selectable marker in biotechnology and enters the environment via laboratory discharges and the release of transgenic organisms. Such releases may provide opportunities for recombination in competent environmental bacteria. The retrieved GenBank sequences were grouped in three datasets comprising river water samples, duck pathogens and full-length variants from various bacterial genomes and plasmids. Analysis for recombination in these datasets was performed with the Recombination Detection Program (RDP4), and the Genetic Algorithm for Recombination Detection (GARD). From a total of 89 homologous sequences, 83% showed 99-100% sequence identity with aph(3')-IIa originally described as part of transposon Tn5. Fifty one were unique sequence variants eligible for recombination analysis. Only a single recombination event was identified with high confidence and indicated the involvement of aph(3')-IIa in the formation of a mosaic gene located on a plasmid of environmental origin in the multi-resistant isolate Pseudomonas aeruginosa PA96. The available data suggest that aph(3')-IIa is not an archetypical mosaic gene as the divergence between the described sequence variants and the number of detectable recombination events is low. This is in contrast to the numerous mosaic alleles reported for certain penicillin or tetracycline resistance determinants.

RESUMEN

In our recent article "In vivo evolution of metabolic pathways by homeologous recombination in mitotic cells" we proposed a useful alternative to directed evolution methods that permits the generation of yeast cell libraries containing recombinant metabolic pathways from counterpart genes. The methodology was applied to generate single mosaic genes and intragenic mosaic pathways. We used flavonoid metabolism genes as a working model to assembly and express evolved pathways in DNA repair deficient cells. The present commentary revises the principles of gene and pathway mosaicism and explores the scope and perspectives of our results as an additional tool for synthetic biology.

Asunto(s)

Evolución Molecular Dirigida/métodos , Recombinación Homóloga , Ingeniería Metabólica/métodos , Mitosis/genética , Saccharomyces cerevisiae

RESUMEN

OBJECTIVES: To screen the ftsI gene sequences obtained from clinical isolates of non-typeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus for the presence of mosaic ftsI gene structures, and to evaluate the role of inter-species recombination of the ftsI gene in the formation and distribution of resistant ftsI genes. METHODS: The ftsI genes of 100 Haemophilus isolates comprising genetically defined ß-lactamase-negative ampicillin-susceptible (gBLNAS), ß-lactamase-positive ampicillin-resistant (gBLPAR), ß-lactamase-negative ampicillin-resistant (gBLNAR) and ß-lactamase-positive amoxicillin/clavulanate-resistant (gBLPACR) isolates of NTHi (n = 50) and H. haemolyticus (n = 50) were analysed in this study. Both the flanking regions and the full-length ftsI gene sequences of all study isolates were screened for mosaic structures using H. influenzae Rd and H. haemolyticus ATCC 33390 as reference parental sequences, and bioinformatics methods were used for recombination analysis using SimPlot. RESULTS: Of the 100 clinical isolates analysed 34% (34/100) harboured mosaic ftsI gene structures containing distinct ftsI gene fragments similar to both reference parental sequences. The inter-species recombination events were exclusively encountered in the ftsI gene of gBLNAR/gBLPACR isolates of both NTHi and H. haemolyticus, and were always associated with the formation of a mosaic fragment at the 3' end of the ftsI gene. There was no evidence supporting horizontal gene transfer (HGT) involving the entire ftsI gene among the clinical isolates in vivo. CONCLUSIONS: We provide evidence for the HGT and inter-species recombination of the ftsI gene among gBLNAR/gBLPACR isolates of NTHi and H. haemolyticus in a clinical setting, highlighting the importance of recombination of the ftsI gene in the emergence of altered penicillin-binding protein 3 and BLNAR-mediated resistance.

Asunto(s)

Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/genética , Haemophilus/efectos de los fármacos , Haemophilus/genética , Resistencia a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/genética , Recombinación Genética , Infecciones por Haemophilus/microbiología , Humanos

RESUMEN

Natural transformation can lead to exchange of DNA between taxonomically diverse bacteria. In the case of chromosomal DNA, homology-based recombination with the recipient genome is usually necessary for heritable stability. In our recent study, we have shown that natural transformation can promote the transfer of transposons, IS elements, and integrons and gene cassettes, largely independent of the genetic relationship between the donor and recipient bacteria. Additional results from our study suggest that natural transformation with species-foreign DNA might result in the uptake of a wide range of DNA fragments; leading to changes in the antimicrobial susceptibility profile and contributing to the generation of antimicrobial resistance in bacteria.