The B subunit of Escherichia coli heat labile enterotoxin (LT-B) is a potent oral immunogen with potential for use as a vaccine, a carrier molecule to deliver antigens to gut-associated lymphoid tissues, and possibly an adjuvant to make coadministered vaccines more effective. LT-B produced in plants was shown to be functional and immunogenic in animals and humans. In this work, we show that maize-derived LT-B is strongly associated with starch granules in endosperm. Using immunogold labeling͞electron microscopy, cell fractionation, and protein analysis techniques, we observed that LT-B protein could be detected both internally and externally in starch granules. This strong association confers an effective copurification of the antigen with the starch fraction of maize kernels, thermostability desirable in maize processing, and resistance to peptic degradation in simulated gastric fluid digests, an important attribute for an orally delivered antigen.LT-B ͉ starch localization

Salmonella enterica is one of the most important foodborne pathogens around the world. In the last years, S . enterica serovar Infantis has become an important emerging pathogen in many countries, often as multidrug resistant clones. To understand the importance of S. enterica in the broiler industry in Ecuador, we performed a study based on phenotypic and WGS data of isolates from poultry farms, chicken carcasses and humans. We showed a high prevalence of S. enterica in poultry farms (41.4%) and chicken carcasses (55.5%), but a low prevalence (1.98%) in human samples. S . Infantis was shown to be the most prevalent serovar with a 98.2, 97.8, and 50% in farms, foods, and humans, respectively, presenting multidrug resistant patterns. All sequenced S . Infantis isolates belonged to ST32. For the first time, a pESI-related megaplasmid was identified in Ecuadorian samples. This plasmid contains genes of antimicrobial resistance, virulence factors, and environmental stress tolerance. Genomic analysis showed a low divergence of S . Infantis strains in the three analyzed components. The results from this study provide important information about genetic elements that may help understand the molecular epidemiology of S . Infantis in Ecuador.

Mapping of high-throughput sequencing (HTS) reads to a single arbitrary reference genome is a frequently used approach in microbial genomics. However, the choice of a reference may represent a source of errors that may affect subsequent analyses such as the detection of single nucleotide polymorphisms (SNPs) and phylogenetic inference. In this work, we evaluated the effect of reference choice on short-read sequence data from five clinically and epidemiologically relevant bacteria (Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Pseudomonas aeruginosa and Serratia marcescens). Publicly available whole-genome assemblies encompassing the genomic diversity of these species were selected as reference sequences, and read alignment statistics, SNP calling, recombination rates, dN/dS ratios, and phylogenetic trees were evaluated depending on the mapping reference. The choice of different reference genomes proved to have an impact on almost all the parameters considered in the five species. In addition, these biases had potential epidemiological implications such as including/excluding isolates of particular clades and the estimation of genetic distances. These findings suggest that the single reference approach might introduce systematic errors during mapping that affect subsequent analyses, particularly for data sets with isolates from genetically diverse backgrounds. In any case, exploring the effects of different references on the final conclusions is highly recommended.

Human Papillomavirus (HPV) is the most common sexually transmitted infection worldwide and it is responsible for most cases of uterine cancer. In Ecuador there is limited information about HPV types (and variants) in cancerous lesions; however, identifying the type-specific HPV prevalence in cervical lesions of women living in Ecuador is important to better predict the impact of HPV prophylactic vaccination in this country. We studied the prevalence of HPV types in cervical cancerous or precancerous lesions from 164 Ecuadorian women and found that 86.0% were HPV positive. The most common types were HPV16 (41.8%) and HPV58 (30.5%). Interestingly, HPV18 was detected only in 2.8% of the HPV-positive samples. Fifteen DNA sequences (genes E6 and L1) from 16 samples positive for HPV16 belonged to the European lineage, considered one of the least carcinogenic lineages, and 1 (6.25%) to the Asian-American lineage. Similar analysis in 12 HPV58 positive samples showed that 10 (83.3%) sequences grouped in sublineage A2, which belongs to the oldest HPV58 lineage, 1 belonged to A3 and 1 to lineage C. This study suggests that the currently used HPV vaccines (bivalent and tetravalent) may have lower effectiveness in Ecuador than in other geographic locations where HPV18 is more prevalent.

16 Mapping of high-throughput sequencing (HTS) reads to a single arbitrary reference genome is a 17 frequently used approach in microbial genomics. However, the choice of a reference may represent a 18 source of errors that may affect subsequent analyses such as the detection of single nucleotide 19 polymorphisms (SNPs) and phylogenetic inference. In this work, we evaluated the effect of reference 20 choice on short-read sequence data from five clinically and epidemiologically relevant bacteria 21 (Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Pseudomonas aeruginosa 22 and Serratia marcescens). Publicly available whole-genome assemblies encompassing the genomic 23 diversity of these species were selected as reference sequences, and read alignment statistics, SNP 24 calling, recombination rates, dN/dS ratios, and phylogenetic trees were evaluated depending on the 25 mapping reference. The choice of different reference genomes proved to have an impact on almost all 26 the parameters considered in the five species. In addition, these biases had potential epidemiological 27 implications such as including/excluding isolates of particular clades and the estimation of genetic 28 distances. These findings suggest that the single reference approach might introduce systematic errors 29 during mapping that affect subsequent analyses, particularly for data sets with isolates from 30 genetically diverse backgrounds. In any case, exploring the effects of different references on the final 31 conclusions is highly recommended. 32 33 Author summary 34 Mapping consists in the alignment of reads (i.e., DNA fragments) obtained through high-throughput 35 genome sequencing to a previously assembled reference sequence. It is a common practice in genomic 36 studies to use a single reference for mapping, usually the 'reference genome' of a species -a high-37 quality assembly. However, the selection of an optimal reference is hindered by intrinsic intra-species 38 genetic variability, particularly in bacteria. Biases/errors due to reference choice for mapping in 39 bacteria have been identified. These are mainly originated in alignment errors due to genetic 40 differences between the reference genome and the read sequences. Eventually, they could lead to 41 misidentification of variants and biased reconstruction of phylogenetic trees (which reflect ancestry 42 between different bacterial lineages). However, a systematic work on the effects of reference choice 43 in different bacterial species is still missing, particularly regarding its impact on phylogenies. This 44 work intended to fill that gap. The impact of reference choice has proved to be pervasive in the five 45 bacterial species that we have studied and, in some cases, alterations in phylogenetic trees could lead 46 to incorrect epidemiological inferences. Hence, the use of different reference genomes may be 47 prescriptive to assess the potential biases of mapping. 48 49 Introduction 50 The development and increasing availability of high-throughput sequen...

Our findings strongly suggest that alcohol-free mouthwash solution intrinsically contaminated with B. cepacia was the source of these colonizations and infections involving adults in the ICU.

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