Genotypic and phenotypic characteristics of Cronobacter species, with particular attention to the newly reclassified species C. helveticus, C. pulveris, and C. zurichensis [forthcoming]

In 2013, Enterobacter helveticus, E. pulveris and E. turicensis, were reclassified as Cronobacter helveticus, C. pulveris and C. zurichensis, respectively. Previously these species had been used as negative controls for some Cronobacter detection assays. This study examined cultural, biochemical and molecular Cronobacter detection and identification assays, with emphasis on the new species. Additionally, 32 Cronobacter genomes were examined for the presence of PCR target genes using the BLAST function of the online Cronobacter BIGSdb facility. The results of the cultural methods varied and no single medium was able to correctly detect all Cronobacter spp. Since the supporting databases have not been updated to include the Cronobacter genus, Enterobacter sakazakii was returned for four strains of the newly reclassified species with ID32E and none with API 20E. PCR probes targeting rpoB and ompA could not correctly identify the new Cronobacter spp., due to primer specificity or absent target genes. As neonates have been identified as a high-risk group for infection, international standards require the absence of all Cronobacter species in powdered infant formula. However, many conventional detection methods cannot correctly identify the newly recognized species. Conversely, DNA sequence-based methods can adapt to taxonomic revisions and will likely become more common

Multilocus sequence typing of Cronobacter spp. from powdered infant formula and milk powder production factories

This study applied the Cronobacter spp. multilocus sequence typing (MLST) scheme to three strain collections, then known as Enterobacter sakazakii, which had been isolated between 1988 and 2009 from 14 countries. The results revealed the predominance (85%) of C. 29 sakazakii (72 strains) in all three collections. The remaining strains were C. turicensis (10%), C. malonaticus (4%), and C. muytjensii (1%). No strains of C. dublinensis, C. universalis or C. condimenti were identified. Twenty-one out of seventy two C. sakazakii strains were in the clinically significant ST4 clonal complex, and were found in all three strain collections. These results confirm C. sakazakii ST4 is one of the predominant clonal complexes over the past 20 years in several parts of the world. Further understanding of the ecosystem and sources of the organism may be used for the development of improved intervention strategies in the dairy industry

In-vitro investigation of biofilm-specific resistance and virulence of biofilm-forming uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the primary etiologic agent of urinary tract infections (UTIs). This study aimed to investigate the difference in antimicrobial susceptibility of UPEC isolates in the planktonic and biofilm states. Important virulence factors were also evaluated. The minimum inhibitory concentrations (MICs) were determined and recorded as 0.5-64 μg/ ml for amikacin, 0.5-64 μg/ ml for cefotaxime, 0.25-64 μg/ ml for cefepime, 0.25-16 μg/ ml for meropenem, and 0.125-32 μg/ ml for ciprofloxacin. Biofilm-specific resistance was assessed using the minimum biofilm eradication concentration (MBEC). The obtained results for MBEC were: 8-512 μg/ ml for amikacin, 32-512 μg/ ml for cefotaxime, 8-512 μg/ ml for cefepime, 4-256 μg/ ml for meropenem, and 4-128 μg/ ml for ciprofloxacin. The virulence factors were evaluated using suitable phenotypic techniques. Our findings revealed a significant enhancement in the antimicrobial resistance after biofilm formation. The MBEC values were higher than the MIC values by 2-128 folds for amikacin, 2-256 folds for cefotaxime, 2-64 folds for cefepime, 8-128 folds for meropenem, and 4-128 folds for ciprofloxacin. The swimming and swarming motilities demonstrated a significant positive correlation (rs = 0.506, P< 0.001). Protease production analysis revealed a large variation, with the weak biofilm-producing isolates EW2 and EW15 displaying the largest zone diameters of 39 mm and 33 mm; respectively. We have also evaluated the distribution and levels of siderophore production, which were significantly associated with meropenem resistance. Finally, this study underscores the importance of considering biofilm formation in UPEC treatment and emphasizes the need for therapeutics targeting these biofilms

Capsular profiling of the Cronobacter genus and the association of specific Cronobacter sakazakii and C. malonaticus capsule types with neonatal meningitis and necrotizing enterocolitis

Background: Cronobacter sakazakii and C. malonaticus can cause serious diseases especially in infants where they are associated with rare but fatal neonatal infections such as meningitis and necrotising enterocolitis. Methods: This study used 104 whole genome sequenced strains, covering all seven species in the genus, to analyse capsule associated clusters of genes involved in the biosynthesis of the O-antigen, colanic acid, bacterial cellulose, enterobacterial common antigen (ECA), and a previously uncharacterised K-antigen. Results: Phylogeny of the gnd and galF genes flanking the O-antigen region enabled the defining of 38 subgroups which are potential serotypes. Two variants of the colanic acid synthesis gene cluster (CA1 and CA2) were found which differed with the absence of galE in CA2. Cellulose (bcs genes) were present in all species, but were absent in C. sakazakii sequence type (ST) 13 and clonal complex (CC) 100 strains. The ECA locus was found in all strains. The K-antigen capsular polysaccharide Region 1 (kpsEDCS) and Region 3 (kpsMT) genes were found in all Cronobacter strains. The highly variable Region 2 genes were assigned to 2 homology groups (K1 and K2). C. sakazakii and C. malonaticus isolates with capsular type [K2:CA2:Cell+] were associated with neonatal meningitis and necrotizing enterocolitis. Other capsular types were less associated with clinical infections. Conclusion: This study proposes a new capsular typing scheme which identifies a possible important virulence trait associated with severe neonatal infections. The various capsular polysaccharide structures warrant further investigation as they could be relevant to macrophage survival, desiccation resistance, environmental survival, and biofilm formation in the hospital environment, including neonatal enteral feeding tubes

Recent advances in the prevention and management of infections in children undergoing treatment for cancer

A major consequence of the intensive multi-modal chemotherapy commonly used to treat malignancies in childhood is life-threatening infection, frequently during periods of profound neutropenia. Recent advances have been made in all areas of management, from trying to prevent infection to getting patients off antimicrobials and home again in the shortest, safest way. Potential avenues of further research are outlined for readers to be aware of in the next few years

Linkage Mapping of Stem Saccharification Digestibility in Rice

Rice is the staple food of almost half of the world population, and in excess 90% of it is grown and consumed in Asia, but the disposal of rice straw poses a problem for farmers, who often burn it in the fields, causing health and environmental problems. However, with increased focus on the development of sustainable biofuel production, rice straw has been recognized as a potential feedstock for non-food derived biofuel production. Currently, the commercial realization of rice as a biofuel feedstock is constrained by the high cost of industrial saccharification processes needed to release sugar for fermentation. This study is focused on the alteration of lignin content, and cell wall chemotypes and structures, and their effects on the saccharification potential of rice lignocellulosic biomass. A recombinant inbred lines (RILs) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 271 molecular markers for quantitative trait SNP (QTS) analyses was used. After association analysis of 271 markers for saccharification potential, 1 locus and 4 pairs of epistatic loci were found to contribute to the enzymatic digestibility phenotype, and an inverse relationship between reducing sugar and lignin content in these recombinant inbred lines was identified. As a result of QTS analyses, several cell-wall associated candidate genes are proposed that may be useful for marker-assisted breeding and may aid breeders to produce potential high saccharification rice varieties

The R2R3-MYB Transcription Factor Gene Family in Maize

MYB proteins comprise a large family of plant transcription factors, members of which perform a variety of functions in plant biological processes. To date, no genome-wide characterization of this gene family has been conducted in maize (Zea mays). In the present study, we performed a comprehensive computational analysis, to yield a complete overview of the R2R3-MYB gene family in maize, including the phylogeny, expression patterns, and also its structural and functional characteristics. The MYB gene structure in maize and Arabidopsis were highly conserved, indicating that they were originally compact in size. Subgroup-specific conserved motifs outside the MYB domain may reflect functional conservation. The genome distribution strongly supports the hypothesis that segmental and tandem duplication contribute to the expansion of maize MYB genes. We also performed an updated and comprehensive classification of the R2R3-MYB gene families in maize and other plant species. The result revealed that the functions were conserved between maize MYB genes and their putative orthologs, demonstrating the origin and evolutionary diversification of plant MYB genes. Species-specific groups/subgroups may evolve or be lost during evolution, resulting in functional divergence. Expression profile study indicated that maize R2R3-MYB genes exhibit a variety of expression patterns, suggesting diverse functions. Furthermore, computational prediction potential targets of maize microRNAs (miRNAs) revealed that miR159, miR319, and miR160 may be implicated in regulating maize R2R3-MYB genes, suggesting roles of these miRNAs in post-transcriptional regulation and transcription networks. Our comparative analysis of R2R3-MYB genes in maize confirm and extend the sequence and functional characteristics of this gene family, and will facilitate future functional analysis of the MYB gene family in maize

Synergistic combination of cytotoxic chemotherapy and cyclin-dependent kinase 4/6 inhibitors in biliary tract cancers

Background and aims: Biliary tract cancers (BTCs) are uncommon, but highly lethal, gastrointestinal malignancies. Gemcitabine/cisplatin is a standard-of-care systemic therapy, but has a modest impact on survival and harbors toxicities, including myelosuppression, nephropathy, neuropathy, and ototoxicity. Whereas BTCs are characterized by aberrations activating the cyclinD1/cyclin-dependent kinase (CDK)4/6/CDK inhibitor 2a/retinoblastoma pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident preclinical efficacy, and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the antitumor efficacy of CDK4/6 inhibitors by the combination with chemotherapy regimens, but their mechanism of action remains elusive.Approach and results: Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity, and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy attributable to autophagy-induced resistance. Notably, triplet therapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated ribonucleotide reductase catalytic subunit M1 reduction, resulting in sensitization to gemcitabine.Conclusions: As such, these data provide robust preclinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.</p