Statistical Evaluation of Metaproteomics and 16S rRNA Amplicon Sequencing Techniques for Study of Gut Microbiota Establishment in Infants with Cystic Fibrosis.

Newborn screening for cystic fibrosis (CF) can identify affected but asymptomatic infants. The selection of omic technique for gut microbiota study is crucial due to both the small amount of feces available and the low microorganism load. Our aims were to compare the agreement between 16S rRNA amplicon sequencing and metaproteomics by a robust statistical analysis, including both presence and abundance of taxa, to describe the sequential establishment of the gut microbiota during the first year of life in a small size sample (8 infants and 28 fecal samples). The taxonomic assignations by the two techniques were similar, whereas certain discrepancies were observed in the abundance detection, mostly the lower predicted relative abundance of Bifidobacterium and the higher predicted relative abundance of certain Firmicutes and Proteobacteria by amplicon sequencing. During the first months of life, the CF gut microbiota is characterized by a significant enrichment of Ruminococcus gnavus, the expression of certain virulent bacterial traits, and the detection of human inflammation-related proteins. Metaproteomics provides information on composition and functionality, as well as data on host-microbiome interactions. Its strength is the identification and quantification of Actinobacteria and certain classes of Firmicutes, but alpha diversity indices are not comparable to those of amplicon sequencing. Both techniques detected an aberrant microbiota in our small cohort of infants with CF during their first year of life, dominated by the enrichment of R. gnavus within a human inflammatory environment. IMPORTANCE In recent years, some techniques have been incorporated for the study of microbial ecosystems, being 16S rRNA gene sequencing being the most widely used. Metaproteomics provides the advantage of identifying the interaction between microorganisms and human cells, but the available databases are less extensive as well as imprecise. Few studies compare the statistical differences between the two techniques to define the composition of an ecosystem. Our work shows that the two methods are comparable in terms of microorganism identification but provide different results in alpha diversity analysis. On the other hand, we have studied newborns with cystic fibrosis, for whom we have described the establishment of an intestinal ecosystem marked by the inflammatory response of the host and the enrichment of Ruminococcus gnavus.

Strain-specific predation of Bdellovibrio bacteriovorus on Pseudomonas aeruginosa with a higher range for cystic fibrosis than for bacteremia isolates.

This work aimed to evaluate the predatory activity of Bdellovibrio bacteriovorus 109J on clinical isolates of Pseudomonas aeruginosa selected from well-characterized collections of cystic fibrosis (CF) lung colonization (n = 30) and bloodstream infections (BSI) (n = 48) including strains selected by genetic lineage (frequent and rare sequence types), antibiotic resistance phenotype (susceptible and multidrug-resistant isolates), and colony phenotype (mucoid and non-mucoid isolates). The intraspecies predation range (I-PR) was defined as the proportion of susceptible strains within the entire collection. In contrast, the predation efficiency (PE) is the ratio of viable prey cells remaining after predation compared to the initial inoculum. I-PR was significantly higher for CF (67%) than for BSI P. aeruginosa isolates (35%) probably related to an environmental origin of CF strains whereas invasive strains are more adapted to humans. I-PR correlation with bacterial features such as mucoid morphotype, genetic background, or antibiotic susceptibility profile was not detected. To test the possibility of increasing I-PR of BSI isolates, a polyhydroxyalkanoate depolymerase deficient B. bacteriovorus bd2637 mutant was used. Global median I-PR and PE values remained constant for both predators, but 31.2% of 109J-resistant isolates were susceptible to the mutant, and 22.9% of 109J-susceptible isolates showed resistance to predation by the mutant, pointing to a predator-prey specificity process. The potential use of predators in the clinical setting should be based on the determination of the I-PR for each species, and the PE of each particular target strain.

Epidermis as a Platform for Bacterial Transmission.

The epidermis constitutes a continuous external layer covering the body, offering protection against bacteria, the most abundant living organisms that come into contact with this barrier. The epidermis is heavily colonized by commensal bacterial organisms that help protect against pathogenic bacteria. The highly regulated and dynamic interaction between the epidermis and commensals involves the host's production of nutritional factors promoting bacterial growth together to chemical and immunological bacterial inhibitors. Signal trafficking ensures the system's homeostasis; conditions that favor colonization by pathogens frequently foster commensal growth, thereby increasing the bacterial population size and inducing the skin's antibacterial response, eliminating the pathogens and re-establishing the normal density of commensals. The microecological conditions of the epidermis favors Gram-positive organisms and are unsuitable for long-term Gram-negative colonization. However, the epidermis acts as the most important host-to-host transmission platform for bacteria, including those that colonize human mucous membranes. Bacteria are frequently shared by relatives, partners, and coworkers. The epidermal bacterial transmission platform of healthcare workers and visitors can contaminate hospitalized patients, eventually contributing to cross-infections. Epidermal transmission occurs mostly via the hands and particularly through fingers. The three-dimensional physical structure of the epidermis, particularly the fingertips, which have frictional ridges, multiplies the possibilities for bacterial adhesion and release. Research into the biology of bacterial transmission via the hands is still in its infancy; however, tribology, the science of interacting surfaces in relative motion, including friction, wear and lubrication, will certainly be an important part of it. Experiments on finger-to-finger transmission of microorganisms have shown significant interindividual differences in the ability to transmit microorganisms, presumably due to genetics, age, sex, and the gland density, which determines the physical, chemical, adhesive, nutritional, and immunological status of the epidermal surface. These studies are needed to optimize interventions and strategies for preventing the hand transmission of microorganisms.

Portación nasal, antibiotipo y genotipo de cepas de Staphylococcus aureus aisladas en estudiantes de Medicina y de Enfermería Campus San Felipe, Universidad de Valparaíso, Chile, durante el año 2017 / Nasal carriage, antibiotype and genotype of isolated Staphylococcus aureus from Medicine and Nursing students of Campus San Felipe, University of Valparaiso, Chile, during 2017

INTRODUCCIÓN. Staphylococcus aureus es parte de la microbiota nasal en 20-30% de la población general, colonización que constituye un reservorio para su transmisión, lo que es preocupante en cepas resistentes a meticilina (SARM). OBJETIVO: Determinar la prevalencia de S. aureus en estudiantes de Medicina y Enfermería del Campus San Felipe y caracterizar sus aislamientos. MATERIAL Y MÉTODOS: El 2017 se midió la portación nasal a 225 estudiantes, a las cepas aisladas se le analizó su antibiotipo por difusión en agar, la relación clonal por electroforesis de campo pulsado y MLST. En SARM se determinó el cassette SCCmec y gen de la leucocidina de Panton-Valentine. RESULTADOS: 61 estudiantes portaron S. aureus (27,1%) incluyendo dos cepas SARM (0,9%). Staphylococcus aureus mostró resistencia a penicilina (75%), eritromicina (14%) y clindamicina (10%), cloranfenicol (1,6%) y levofloxacina, oxacilina, cefoxitina (3,3%). Se diferenciaron diecinueve pulsotipos y el secuenciotipo coincidió con complejos clonales descritos a nivel mundial en portadores de S. aureus: CC30, CC8, CC97, CC15, CC22 y CC1. Las dos cepas SARM correspondieron con los clones chileno/cordobés y USA100NY/J, ambas del CC5. CONCLUSIÓN: La portación nasal de S. aureus y SARM en los estudiantes coincidió con la portación en la población general y las cepas sensibles a meticilina mostraron diversidad clonal y alta susceptibilidad antimicrobiana, exceptuando a penicilina.

BACKGROUND: Staphylococcus aureus is part of the nasal microbiota in 20-30% of the population. This colonization is also a reservoir for its dissemination, which is worrying in the case of strains with resistance to methicillin (MRSA). AIM: To determine S. aureus nasal carriage in nursing and medical students of San Felipe Campus and characterize theirs isolates. METHODS: During 2017, nasal swabs were taken from 225 students and seeded in salt manitol agar. Antibiotypes were determined by agar diffusion and the genetic clonality was assessed by PFGE and MLST in isolated S. aureus. SCCmec cassette and Panton-Valentine leukocidin gene (pvl) presence were determined in the MRSA isolates. RESULTS: 61 students carried S. aureus (27.1%) including two MRSA strains (0.9%). S. aureus showed resistance to penicillin (75%), erythromycin (14%) and clindamycin (10%), chloramphenicol (1.6%) and levofloxacin, oxacillin, cefoxitin (3.3%). Nineteen PFGE-types were differentiated, and their sequence-types coincided with main clonal complexes described in S. aureus carriers from different places worldwide: CC30, CC8, CC97, CC15, CC22 and CC1. MRSA strains belonged to CC5 and they corresponded to the Chilean/Cordobes and USA100NY/J clones. CONCLUSION: Nasal carriage of S. aureus and MRSA in students, coincided with the general population and sensitive-methicillin strains showed clonal diversity and high antimicrobial susceptibility except for penicillin.

Machine Learning Study in Caries Markers in Oral Microbiota from Monozygotic Twin Children.

In recent years, the etiology of caries has evolved from a simplistic infectious perspective based on Streptococcus mutans and/or Lactobacillus activity, to a multifactorial disease involving a complex oral microbiota, the human genetic background and the environment. The aim of this work was to identify bacterial markers associated with early caries using massive 16S rDNA. To minimize the other factors, the composition of the oral microbiota of twins in which only one of them had caries was compared with their healthy sibling. Twenty-one monozygotic twin pairs without a previous diagnosis of caries were recruited in the context of their orthodontic treatment and divided into two categories: (1) caries group in which only one of the twins had caries; and (2) control group in which neither of the twins had caries. Each participant contributed a single oral lavage sample in which the bacterial composition was determined by 16S rDNA amplification and further high-throughput sequencing. Data analysis included statistical comparison of alpha and beta diversity, as well as differential taxa abundance between groups. Our results show that twins of the control group have a closer bacterial composition than those from the caries group. However, statistical differences were not detected and we were unable to find any particular bacterial marker by 16S rDNA high-throughput sequencing that could be useful for prevention strategies. Although these results should be validated in a larger population, including children from other places or ethnicities, we conclude that the occurrence of caries is not related to the increase of any particular bacterial population.

[Nasal carriage, antibiotype and genotype of isolated Staphylococcus aureus from Medicine and Nursing students of Campus San Felipe, University of Valparaiso, Chile, during 2017]. / Portación nasal, antibiotipo y genotipo de cepas de Staphylococcus aureus aisladas en estudiantes de Medicina y de Enfermería Campus San Felipe, Universidad de Valparaíso, Chile, durante el año 2017.

BACKGROUND: Staphylococcus aureus is part of the nasal microbiota in 20-30% of the population. This colonization is also a reservoir for its dissemination, which is worrying in the case of strains with resistance to methicillin (MRSA). AIM: To determine S. aureus nasal carriage in nursing and medical students of San Felipe Campus and characterize theirs isolates. METHODS: During 2017, nasal swabs were taken from 225 students and seeded in salt manitol agar. Antibiotypes were determined by agar diffusion and the genetic clonality was assessed by PFGE and MLST in isolated S. aureus. SCCmec cassette and Panton-Valentine leukocidin gene (pvl) presence were determined in the MRSA isolates. RESULTS: 61 students carried S. aureus (27.1%) including two MRSA strains (0.9%). S. aureus showed resistance to penicillin (75%), erythromycin (14%) and clindamycin (10%), chloramphenicol (1.6%) and levofloxacin, oxacillin, cefoxitin (3.3%). Nineteen PFGE-types were differentiated, and their sequence-types coincided with main clonal complexes described in S. aureus carriers from different places worldwide: CC30, CC8, CC97, CC15, CC22 and CC1. MRSA strains belonged to CC5 and they corresponded to the Chilean/Cordobes and USA100NY/J clones. CONCLUSION: Nasal carriage of S. aureus and MRSA in students, coincided with the general population and sensitive-methicillin strains showed clonal diversity and high antimicrobial susceptibility except for penicillin.

Genomics of Serratia marcescens Isolates Causing Outbreaks in the Same Pediatric Unit 47 Years Apart: Position in an Updated Phylogeny of the Species.

The first documented nosocomial outbreak caused by Serratia marcescens in Spain occurred in 1969 at the neonatal intensive care unit (NICU) of the tertiary La Paz Children's Hospital in Madrid, Spain, and based on the available phenotyping techniques at this time, it was considered as a monoclonal outbreak. Only 47 years later, another S. marcescens outbreak of an equivalent dimension occurred at the same NICU. The aim of the present study was to study isolates from these historical and contemporary outbreaks by phenotypic analysis and whole-genome sequencing techniques and to position these strains along with 444 publicly available S. marcescens genomes, separately comparing core genome and accessory genome contents. Clades inferred by both approaches showed high correlation, indicating that core and accessory genomes seem to evolve in the same manner for S. marcescens. Nine S. marcescens clusters were identified, and isolates were grouped in two of them according to sampling year. One exception was isolate 13F-69, the most genetically distant strain, located in a different cluster. Categorical functions in the annotated accessory genes of both collections were preserved among all isolates. No significant differences in frequency of insertion sequences in historical (0.18-0.20)-excluding the outlier strain-versus contemporary isolates (0.11-0.19) were found despite the expected resting effect. The most dissimilar isolate, 13F-69, contains a highly preserved plasmid previously described in Bordetella bronchiseptica. This strain exhibited a few antibiotic resistance genes not resulting in a resistant phenotype, suggesting the value of gene down expression in adaptation to long-term starvation.

Influence of a Serratia marcescens outbreak on the gut microbiota establishment process in low-weight preterm neonates.

Adequate gut microbiota establishment is important for lifelong health. The aim was to sequentially analyze the gut microbiota establishment in low-birth-weight preterm neonates admitted to a single neonatal intensive care unit during their first 3 weeks of life, comparing two epidemiological scenarios. Seven control infants were recruited, and another 12 during a severe S. marcescens outbreak. Meconium and feces from days 7, 14, and 21 of life were collected. Gut microbiota composition was determined by 16S rDNA massive sequencing. Cultivable isolates were genotyped by pulsed-field gel electrophoresis, with four S. marcescens submitted for whole-genome sequencing. The expected bacterial ecosystem expansion after birth is delayed, possibly related to antibiotic exposure. The Proteobacteria phylum dominates, although with marked interindividual variability. The outbreak group considerably differed from the control group, with higher densities of Escherichia coli and Serratia to the detriment of Enterococcus and other Firmicutes. Curiously, obligate predators were only detected in meconium and at very low concentrations. Genotyping of cultivable bacteria demonstrated the high bacterial horizontal transmission rate that was confirmed with whole-genome sequencing for S. marcescens. Preterm infants admitted at NICU are initially colonized by homogeneous microbial communities, most of them from the nosocomial environment, which subsequently evolve according to the individual conditions. Our results demonstrate the hospital epidemiology pressure, particularly during outbreak situations, on the gut microbiota establishing process.