Prevalence and Population Diversity of Listeria monocytogenes Isolated from Dairy Cattle Farms in the Cantabria Region of Spain

Listeria monocytogenes is an opportunistic pathogen that is widely distributed in the environment. Here we show the prevalence and transmission of L. monocytogenes in dairy farms in the Cantabria region, on the northern coast of Spain. A total of 424 samples was collected from 14 dairy farms (5 organic and 9 conventional) and 211 L. monocytogenes isolates were recovered following conventional microbiological methods. There were no statistically significant differences in antimicrobial resistance ratios between organic and conventional farms. A clonal relationship among the isolates was assessed by pulsed field gel electrophoresis (PFGE) analysis and 64 different pulsotypes were obtained. Most isolates (89%, n = 187) were classified as PCR serogroup IVb by using a multiplex PCR assay. In this case, 45 isolates of PCR serogroup IVb were whole genome-sequenced to perform a further analysis at genomic level. In silico MLST analysis showed the presence of 12 sequence types (ST), of which ST1, ST54 and ST666 were the most common. Our data indicate that the environment of cattle farms retains a high incidence of L. monocytogenes, including subtypes involved in human listeriosis reports and outbreaks. This pathogen is shed in the feces and could easily colonize dairy products, as a result of fecal contamination. Effective herd and manure management are needed in order to prevent possible outbreaks.This work was supported by Research Project grants RTA08-099, RTA2008-00080-C02, RTA2014-00045-C03-01 (INIA and FEDER) from the Spanish Ministry of Economy and Competitiveness and RTI2018-098267-R-C31 (INIA and FEDER) from the Spanish Ministry of Science and Innovation.S

Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures

During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.This work was financed by grants BFU2014-55534-C2-1-P from the Spanish Ministry of Economy and Competitiveness and 612146/FP7-ICT- 2013 and 282004/FP7-HEALTH.2011.2.3.1-2 from the European Union Seventh Framework Programme to FC and grant BFU2014-55534-C2-2-P from the Spanish Ministry of Economy and Competitiveness to GM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A degenerate primer MOB typing (DPMT) method to classify gamma-proteobacterial plasmids in clinical and environmental settings

Transmissible plasmids are responsible for the spread of genetic determinants, such as antibiotic resistance or virulence traits, causing a large ecological and epidemiological impact. Transmissible plasmids, either conjugative or mobilizable, have in common the presence of a relaxase gene. Relaxases were previously classified in six protein families according to their phylogeny. Degenerate primers hybridizing to coding sequences of conserved amino acid motifs were designed to amplify related relaxase genes from γ-Proteobacterial plasmids. Specificity and sensitivity of a selected set of 19 primer pairs were first tested using a collection of 33 reference relaxases, representing the diversity of γ-Proteobacterial plasmids. The validated set was then applied to the analysis of two plasmid collections obtained from clinical isolates. The relaxase screening method, which we call "Degenerate Primer MOB Typing" or DPMT, detected not only most known Inc/Rep groups, but also a plethora of plasmids not previously assigned to any Inc group or Rep-type

Simultaneous ethanol and bacterial ice nuclei from sugar beet molasses by a Zymomonas mobilis CP4 mutant expressing the inaZ gene of Pseudomonas syringae in continuous culture

Aim: The aim of this work was to construct a Zymomonas mobilis mutant capable of simultaneous ethanol and ice nuclei production from agricultural by-product such as sugar beet molasses, in steady-state continuous culture. Methods and Results: A sucrose-hypertolerant mutant of Z. mobilis strain CP4, named suc40, capable of growing on 40% (w/v) sucrose medium was isolated following N-methyl-N′-nitro-N-nitrosoguanidine treatment. Plasmid pDS3154 carrying the inaZ gene of Pseudomonas syringae was conjugally transferred and expressed in suc40. The potential for simultaneous ethanol and bacterial ice nuclei production was assessed in steady-state continuous cultures over a range of dilution rates from 0·04 to 0·13 h-1. In addition, the fatty acid and phospholipid profile of the three strains was also investigated. Ethanol production up to 43 g l-1 was achieved at dilution rates below 0·10 h-1 in sugar beet molasses. Ice nucleation activity gradually increased with increasing dilution rate and the greatest activity, -3·4 log (ice nuclei per cell), was observed at the highest dilution rate (0·13 h-1). Both mutant strains displayed a different fatty acid and phospholipid profile compared with the wild-type strain. Conclusions: The ability of the mutant and recombinant plasmid-containing strains to grow on high sugar concentrations and in high osmotic pressure environments (molasses) can be attributed to their phospholipid and fatty acid contents. Significance and Impact of the Study: Taking into account that sugar beet molasses is a low cost agricultural by-product, the simultaneous ethanol and bacterial ice nucleation production achieved under the studied conditions is considered very promising for industrial applications

New Sequence Type ST3449 in Multidrug-Resistant Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient

Pseudomonas aeruginosa is one of the most critical bacterial pathogens associated with chronic infections in cystic fibrosis patients. Here we show the phenotypic and genotypic characterization of five consecutive multidrug-resistant isolates of P. aeruginosa collected during a month from a CF patient with end-stage lung disease and fatal outcome. The isolates exhibited distinct colony morphologies and pigmentation and differences in their capacity to produce biofilm and virulence potential evaluated in larvae of Galleria mellonella. Whole genome-sequencing showed that isolates belonged to a novel sequence type ST3449 and serotype O6. Analysis of their resistome demonstrated the presence of genes blaOXA-396, blaPAO, aph(3’)-IIb, catB, crpP and fosA and new mutations in chromosomal genes conferring resistance to different antipseudomonal antibiotics. Genes exoS, exoT, exoY, toxA, lasI, rhlI and tse1 were among the 220 virulence genes detected. The different phenotypic and genotypic features found reveal the adaptation of clone ST3449 to the CF lung environment by a number of mutations affecting genes related with biofilm formation, quorum sensing and antimicrobial resistance. Most of these mutations are commonly found in CF isolates, which may give us important clues for future development of new drug targets to combat P. aeruginosa chronic infections