Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis.

MexAB-OprM and MexEF-OprN are Pseudomonas aeruginosa efflux pumps involved in the development of antibiotic resistance. Several studies developed with laboratory strains or using a few clinical isolates have reported that the regulation system of MexEF-OprN is involved in the final levels of MexAB-OprM expression. Therefore, this study was aimed to determine the interplay between MexAB-OprM and MexEF-OprN in 90 out of 190 P. aeruginosa clinical isolates with an efflux pump overexpression phenotype. Regarding oprD, 33% (30/90) of isolates displayed relevant modifications (RM) defined as frameshift or premature stop, both related to carbapenem resistance. On the other hand, 33% of the isolates displayed RM in nalC, nalD or mexR, which were significantly associated with multidrug resistance (MDR), non-susceptibility to carbapenems, OprD alterations and strong biofilm production. Meanwhile, the RM in MexS were associated with presence of pigment (p = 0.004). Otherwise, when all the regulators were analysed together, the association between RM in MexAB-OprM regulators and MDR was only significant (p = 0.039) when mexS was the wild type. These data show the modulatory effect of MexEF-OprN on MexAB-OprM in a clinical population of P. aeruginosa. Further studies may contribute to design of novel molecules acting on this interplay to fight against antimicrobial resistance.

Background: Recent studies have shown that mammalian milk represents a continuous supply of commensal bacteria, including enterococci. The objectives of this study were to evaluate the presence of enterococci in milk of different species and to screen them for several genetic and phenotypic traits of clinical significance among enterococci. Results: Samples were obtained from, at least, nine porcine, canine, ovine, feline and human healthy hosts. Enterococci could be isolated, at a concentration of 1.00 × 10² -1.16 × 10³ CFU/ml, from all the porcine samples and, also from 85, 50, 25 and 25% of the human, canine, feline and ovine ones, respectively. They were identified as Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus durans. Among the 120 initial enterococcal isolates, 36 were selected on the basis of their different PFGE profiles and further characterized. MLST analysis revealed a wide diversity of STs among the E. faecalis and E. faecium strains, including some frequently associated to hospital infections and novel STs. All the E. faecalis strains possessed some of the potential virulence determinants (cad, ccf, cob, cpd, efaA _fs , agg2, gelE, cylA, esp _fs ) assayed while the E. faecium ones only harboured the efaA _fm gene. All the tested strains were susceptible to tigecycline, linezolid and vancomycin, and produced tyramine. Their susceptibility to the rest of the antimicrobials and their ability to produce other biogenic amines varied depending on the strain. Enterococci strains isolated from porcine samples showed the widest spectrum of antibiotic resistance. Conclusions: Enterococci isolated from milk of different mammals showed a great genetic diversity. The wide distribution of virulence genes and/or antibiotic resistance among the E. faecalis and E. faecium isolates indicates that they can constitute a reservoir of such traits and a risk to animal and human health.