Aetiology of potentially pathogenic bacteria from neonatal feeding tubes

Recently, the incidence of neonatal infections, particularly in those born with low birth weight (< 2000g) has increased due to Enterobacteriaceae including Escherichia coli, Klebsiella and Serratia spp., and other Gram-positive bacteria such as Enterococcus spp. These are known to be responsible for major neonatal intensive care unit (NICU) infections. Mortality among neonates is attributed to infectious causes, preterm birth complications, intrapartum-related complications, sepsis and meningitis. Therefore, this study aimed: (a) to evaluate the potential risk to neonates posed by ingestion of E. coli and Klebsiella spp, E. hormaechei and E. faecalis either through powdered infant formula, contaminated milk, or by medical equipment, (b) to categorise isolates of these organisms into high, medium and low potential pathogenicity to neonates, and (c) to conduct a longitudinal study to determine whether the same strain colonises both the feeding tube and intestine of a premature baby in the NICU over time and to determine their virulence potential and genetic relationships. This study used, 76 K. pneumoniae strains previously isolated from neonatal feeding tubes, from two Jordanian hospitals from May to Dec 2011 and 36 isolates (E. coli n=14 and Klebsiella n=22) from Queen's Medical Centre and Nottingham City Hospital (QMC & NCH, Nottingham), from neonatal enteral feeding tubes and sepsis cases between 2007 and 2015. In addition, 14 isolates (E. faecalis n= 8 and E. hormaechei n= 6) were collected from four samples (two nasogastric feeding tube and two faecal samples) from a single premature baby at age 6 and 8 weeks of life in the NICU at QMC, Nottingham. The isolates were identified by sequence analysis of the rpoB gene, 16S rDNA and genotyped using pulsed-field gel electrophoresis (XbaI, Spel and SmaI restriction digestion), subsequently profiled using specific PCR probes for virulence genes (K1, 2, and 5, fimbria type, invasion gene and yersiniabactin). Isolates were examined for potential virulence factors including biofilm, capsule production, serum resistance, siderophores, heat resistance, desiccation tolerance and antibiotic resistance. Potential virulence traits were predicted from whole genome sequences. In addition, in vitro tissue culture assays (attachment, invasion and macrophage survival) were used in a comparative study between representative K. pneumoniae strains isolated from EFT Jordanian hospitals and Nottingham NEFTs isolates and sepsis isolates. The K. pneumoniae Jordanian strains clustered into five pulsotypes, pulsotypes 1 and 2 were rpoB allele profile 4, cluster 3 and 4 were rpoB profile 25 and pulsotype 5 was rpoB profile 21. The curli fimbriae and hypermucoviscous phenotype were observed in 10.7 % and 67.9 % of isolates respectively. Capsular serotypes included K1 (17.9%) and K2 (78.6%). All isolates showed resistance to imipenem, meropenim and ceftriaxone. An extended spectrum betalactamase (ESBL) was identified phenotypically in strains with resistance to cefotaxime + clavulanate and cefpodoxime + clavulanate, and most isolates showed susceptibility to ciprofloxacin. All strains had ɣ-haemolytic activity on sheep blood and most showed ßhaemolytic on horse blood. Most isolates were able to form biofilms on plastic surfaces at 25 oC and 37 oC. Most of the strains were able to survive in pH 3.5 for up to 2 hours and tolerated human serum. For the E. coli (QMC) strains, PFGE showed two E. coli strains 2113 and 2114 clustered together, whereas the other strains were all unique. Thirteen E. coli strains from QMC & NCH belonged to phylogenetic group B2 and strain 2255 was group D. The majority of isolates showed ɣ-haemolytic activity on both horse and sheep blood, capsular type K1 and type 1 fimbria were detected. Almost all of the strains were resistant to augmentin, whereas only 7% of the strains were resistant to ceftazidime. ESBL production was identified phenotypically in 99% of the strains with resistance to cefotaxime + clavulanate (CTX+CV) and cefpodoxime + clavulanate (CPD+CV). Furthermore, 77% of the strains were multidrug resistant. All strains were able to form biofilms at 25oC and 37oC. In addition, Klebsiella spp. (QMC) strains revealed eight different rpoB alleles profiles; 1,2,4,10,13,14,15,40. Using PFGE, strains 498 and 500 were clustered together, whereas the other strains were all unique. Phenotypically, all isolates were able to lyse horse erythrocytes, showing ß-haemolytic activity, and were ɣ-haemolytic on sheep blood. Furthermore, most of the strains possessed plasmids and were able to produce cellulose, hyperviscous capsule and biofilm in TSB and infant formula milk (IF). Most strains were able to form capsular material on XLD and IF agar. Only strain 1444 expressed curli fimbriae and produced high levels of biofilm on both media. The great majority of these K. pneumoniae isolates were able to adhere and invade T24, HMBEC and Caco2 cell lines. Unexpectedly, K. pneumoniae strains isolated from FT from Jordan were significantly better at surviving within macrophage (U937) cells than strains isolated from sepsis blood cultures from QMC, Nottingham. The longitudinal study indicated that the E. hormaechei and E. faecalis strains isolated persisted in the baby’s gut across the whole period of study without genotypic and phenotypic changes. Some isolates from feeding tubes and faeces of the baby over time were found to be the same strain based on their sequence type and genomic analysis. All of these strains were shown to have high pathogenic potential

IL4 and IFNalpha generation of dendritic cells reveals great migratory potential and NFkB and cJun expression in IL4DCs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Dendritic cells (DCs) recently revealed as a potent tumor vaccine component, are commonly differentiated from monocytes by cultivation with IL-4 and GM-CSF. Despite the different opinions, the use of IFNalpha can promote DCs differentiation and activation. The aim of this study was to compare the functionality and phenotypic characterization of monocyte-derived DC generated by IL-4 (IL4DC) and IFNalpha (IFNalphaDC) modified protocols. To this aim, we investigated the expression of maturation markers, co-stimulatory molecules, relevant miRNA, cytokine and migratory profiles and the functional ability of these cells to stimulate autologous T cells in vitro. We herein investigated the molecular mechanism underlying the parameters previously described, as the relative expression of NF-kB p65, c-fos and c-jun, transcription factors. Our results demonstrated that IL4DC presented a stable phenotype, an increase in migratory capacity and NF-KB activation, in addition to lower levels of miR-146 a and miR-221. We believe that the IL4DC migratory potential and increase in NFkBp65 expression may be involved in higher IL12 expression and migration, suggesting a preferential activation of TH1 immune responses by IL4DC.428711725Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)INCTSFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP