DNA Repair and Immune Response Pathways Are Deregulated in Melanocyte-Keratinocyte Co-cultures Derived From the Healthy Skin of Familial Melanoma Patients.

Familial melanoma accounts for 10% of cases, being CDKN2A the main high-risk gene. However, the mechanisms underlying melanomagenesis in these cases remain poorly understood. Our aim was to analyze the transcriptome of melanocyte-keratinocyte co-cultures derived from healthy skin from familial melanoma patients vs. controls, to unveil pathways involved in melanoma development in at-risk individuals. Accordingly, primary melanocyte-keratinocyte co-cultures were established from the healthy skin biopsies of 16 unrelated familial melanoma patients (8 CDKN2A mutant, 8 CDKN2A wild-type) and 7 healthy controls. Whole transcriptome was captured using the SurePrint G3 Human Microarray. Transcriptome analyses included: differential gene expression, functional enrichment, and protein-protein interaction (PPI) networks. We identified a gene profile associated with familial melanoma independently of CDKN2A germline status. Functional enrichment analysis of this profile showed a downregulation of pathways related to DNA repair and immune response in familial melanoma (P < 0.05). In addition, the PPI network analysis revealed a network that consisted of double-stranded DNA repair genes (including BRCA1, BRCA2, BRIP1, and FANCA), immune response genes, and regulation of chromosome segregation. The hub gene was BRCA1. In conclusion, the constitutive deregulation of BRCA1 pathway genes and the immune response in healthy skin could be a mechanism related to melanoma risk.

Azithromycin resistance levels and mechanisms in Escherichia coli.

Despite azithromycin being used in some countries to treat infections caused by Gram-negative pathogens, no resistance breakpoint for Escherichia coli exists. The aim of this study was to analyse the levels and mechanisms of azithromycin resistance in E. coli. The presence of chromosomal (rplD, rplV and 23S rRNA) mutations, 10 macrolide resistance genes (MRGs) and efflux pump overexpression was determined in 343 E. coli isolates. Overall, 89 (25.9%) isolates had MICs ≥ 32 mg/L to azithromycin, decreasing to 42 (12.2%) when assayed in the presence of Phe-Arg-ß-Napthylamide, with 35 of these 42 possessing at least one MRG. Efflux pumps played a role in azithromycin resistance affecting the Minimal Inhibitory Concentration (MIC) levels of 91.2% isolates whereas chromosomal alterations seem to have a minimal role. At least one MRG was found in 22.7% of the isolates with mph(A) being the most commonly found gene. The mph(A) gene plays the main role in the development of azithromycin resistance and 93% of the mph(A)-carrying isolates showed a MIC of 32 mg/L. In the absence of a specific resistance breakpoint our results suggest a MIC of 32 mg/L to be considered in order to detect isolates carrying mechanisms able to confer azithromycin resistance.

Resistance to quinolones, cephalosporins and macrolides in Escherichia coli causing bacteraemia in Peruvian children.

OBJECTIVES: To characterise the ß-lactam, quinolone and macrolide resistance levels and mechanisms in 62 Escherichia coli isolates causing bacteraemia in Peruvian children. METHODS: Minimum inhibitory concentrations (MICs) of ciprofloxacin, nalidixic acid (NAL) and azithromycin were determined in the presence and absence of Phe-Arg-ß-naphthylamide. Susceptibility to other 14 antimicrobial agents was also established. Extended-spectrum ß-lactamases (ESBLs) were identified, and mutations in gyrA and parC as well as the presence of transferable mechanisms of quinolone resistance (TMQR) and macrolide resistance (TMMR) were determined. RESULTS: Fifty isolates (80.6%) were multidrug-resistant. High proportions of resistance to ampicillin (93.5%), NAL (66.1%) and trimethoprim/sulfamethoxazole (66.1%) were observed. No isolate showed resistance to carbapenems and only two isolates were resistant to nitrofurantoin. Twenty-seven isolates carried ESBL-encoding genes: 2 blaSHV-12; 13 blaCTX-M-15; 4 blaCTX-M-2; 6 blaCTX-M-65; and 2 non-identified ESBLs. Additionally, 27 blaTEM-1 and 9 blaOXA-1-like genes were detected. All quinolone-resistant isolates showed target mutations, whilst TMQR were present in four isolates. Efflux pumps played a role in constitutive NAL resistance. The association between quinolone resistance and ESBL production was significant (P=0.0011). The mph(A) gene was the most frequent TMMR (16 isolates); msr(A) and erm(B) genes were also detected. Only one TMMR-carrying isolate [presenting mph(A) and erm(B) concomitantly] remained resistant to azithromycin when efflux pumps were inhibited. CONCLUSIONS: A variety of ESBL-encoding genes and widespread of blaCTX-M-15 in Lima has been shown. The role of efflux pumps in azithromycin resistance needs to be further evaluated, as well as effective control of the use of antimicrobial agents.

Virulence factors profiles and ESBL production in Escherichia coli causing bacteremia in Peruvian children.

The presence of 25 virulence genes (VGs), genetic phylogroups, quinolone-resistance and Extended Spectrum ß-lactamase (ESBL)-production was assessed in 65 Escherichia coli isolates from blood cultures in children <5 years in Peru. The most frequent VGs were fimA (89.2%), iutA (83.1%), agn43 (72.3%), iucA (67.7%), and fyuA (49.2%). The isolates belonged to D (47.7%), A (26.1%), B1 (21.5%), and B2 (4.6%) phylogroups. D + B2 isolates presented a high number of fimA, hly, papC, sat, and fyuA genes. Quinolone-susceptible (22 isolates - 33.8%) and ESBL-negative (31 isolates - 47.7%) isolates carried more VGs that their respective counterparts (5.7 vs. 4.7 and 5.3 vs. 4.4 respectively); the frequency of the fyuA, aat, aap, and hly genes significantly differed between quinolone-resistant and quinolone-susceptible isolates. Neonatal sepsis isolates tended to be more quinolone-resistant (P = 0.0697) and ESBL-producers (P = 0.0776). Early-onset neonatal sepsis isolates possessed a high number of VGs (5.2 VGs), especially in neonates of ≤1 day (5.9 VGs).