Raw milk cheeses from Beira Baixa, Portugal-A contributive study for the microbiological hygiene and safety assessment.

Due to specific bacterial microbiota, raw milk cheeses have appreciated sensory properties. However, they may pose a threat to consumer safety due to potential pathogens presence. This study evaluated the microbiological contamination of 98 raw milk cheeses from Beira Baixa, Portugal. Presence and enumeration of Coagulase Positive Staphylococci (CPS), Listeria monocytogenes, Salmonella spp., pathogenic Escherichia coli, and indicator microorganisms (non-pathogenic E. coli and Listeria spp.) was attained. E. coli antimicrobial resistance (AMR) was also evaluated. PCR and/or Whole genome sequencing (WGS) was used to characterize E. coli, Salmonella spp. and L. monocytogenes isolates. Sixteen cheeses (16.3%) were classified as Satisfactory, 59 (60.2%) as Borderline and 23 (23.5%) as Unsatisfactory/Potential Injurious to Health. L. monocytogenes, CPS > 104 cfu g-1, Extraintestinal pathogenic E. coli (ExPEC) and Salmonella spp. were detected in 4.1%, 6.1%, 3.1% and 1.0% of the samples, respectively. Listeria innocua (4.1%) and E. coli > 104 cfu g-1 (16.3%) were also detected. AMR E. coli was detected in 23/98 (23.5%) of the cheese samples, of which two were multidrug resistant. WGS identified genotypes already associated to human disease and Listeria spp. cluster analysis indicated that cheese contamination might be related with noncompliance with Good Hygiene Practices during cheese production.

Multiomics Assessment of Gene Expression in a Clinical Strain of CTX-M-15-Producing ST131 Escherichia coli.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strain C999 was isolated of a Spanish patient with urinary tract infection. Previous genotyping indicated that this strain presented a multidrug-resistance phenotype and carried beta-lactamase genes encoding CTX-M-15, TEM-1, and OXA-1 enzymes. The whole-cell proteome, and the membrane, cytoplasmic, periplasmic and extracellular sub-proteomes of C999 were obtained in this work by two-dimensional gel electrophoresis (2DE) followed by fingerprint sequencing through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). A total of 602 proteins were identified in the different cell fractions, several of which are related to stress response systems, cellular responses, and antibiotic and drug responses, consistent with the multidrug-resistance phenotype. In parallel, whole genome sequencing (WGS) and RNA sequencing (RNA-Seq) was done to identify and quantify the genes present and expressing. The in silico prediction following WGS confirmed our strain as being serotype O25:H4 and sequence type ST131. The presence of proteins related to antibiotic resistance and virulence in an O25:H4-ST131 E. coli clone are serious indicators of the continued threat of antibiotic resistance spread amongst healthcare institutions. On a positive note, a multiomics approach can facilitate surveillance and more detailed characterization of virulent bacterial clones from hospital environments.

Assessment of the load and transcriptional dynamics of Chlamydia trachomatis plasmid according to strains' tissue tropism.

Chlamydia trachomatis maintain a conserved plasmid, which is a primary regulator of chromosomal genes, but there is no experimental evidences associating it with the strains' differential tissue tropism (ocular and genital mucosae, and lymph nodes). We investigated if the number of plasmids per strain correlate with expression profiles of plasmid ORFs and small anti-sense RNAs (sRNAs), and also if these molecular features underlie tropism dissimilarities. We performed absolute and relative qPCR to determine both the plasmid load and expression throughout C. trachomatis development. Our findings suggest that plasmid load (never exceeding 8 copies) is not a function of expression needs and does not reflect tissue tropism. However, for most ORFs, ocular strains presented lower expression than genital or lymphogranuloma venereum (LGV) strains, and ORF6/pgp4 (transcriptional regulator of virulence associated genes) presented the highest mean expression among strains, followed by the virulence factor ORF5/pgp3 (also regulated by ORF6/pgp4). More, the mean expression levels of the sRNA-2 (anti-sense to ORF2/pgp8) were up to 100-fold higher than those of the ORFs, and up to 12-fold higher than that of sRNA-7 (anti-sense to ORF7/pgp5) for the LGV strains. Overall, besides the known regulatory role of C. trachomatis plasmid, its transcriptional dynamics sustains tropism differences.