Lorenza Putignani

Multiplex PCR Allows Rapid and Accurate Diagnosis of Bloodstream Infections in Newborns and Children with Suspected Sepsis

ABSTRACT Sepsis is a major health problem in newborns and children. Early detection of pathogens allows initiation of appropriate antimicrobial therapy that strongly correlates with positive outcomes. Multiplex PCR has the potential to rapidly identify bloodstream infections, compensating for the loss of blood culture sensitivity. In an Italian pediatric hospital, multiplex PCR (the LightCycler SeptiFast test) was compared to routine blood culture with 1,673 samples obtained from 803 children with suspected sepsis; clinical and laboratory information was used to determine the patient infection status. Excluding results attributable to contaminants, SeptiFast showed a sensitivity of 85.0% (95% confidence interval [CI] = 78.7 to 89.7%) and a specificity of 93.5% (95% CI = 92.1 to 94.7%) compared to blood culture. The rate of positive results was significantly higher with SeptiFast (14.6%) than blood culture (10.3%) (P < 0.0001), and the overall positivity rate was 16.1% when the results of both tests were combined. Staphylococcus aureus (11.6%), coagulase-negative staphylococci (CoNS) (29.6%), Pseudomonas aeruginosa (16.5%), and Klebsiella spp. (10.1%) were the most frequently detected. SeptiFast identified 97 additional isolates that blood culture failed to detect (24.7% P. aeruginosa, 23.7% CoNS, 14.4% Klebsiella spp., 14.4% Candida spp.). Among specimens taken from patients receiving antibiotic therapy, we also observed a significantly higher rate of positivity of SeptiFast than blood culture (14.1% versus 6.5%, respectively; P < 0.0001). On the contrary, contaminants were significantly more frequent among blood cultures than SeptiFast (n = 97 [5.8%] versus n = 26 [1.6%]), respectively; P < 0.0001). SeptiFast served as a highly valuable adjunct to conventional blood culture in children, adding diagnostic value and shortening the time to result (TTR) to 6 h.

Global Distribution, Public Health and Clinical Impact of the Protozoan Pathogen Cryptosporidium

Cryptosporidium spp. are coccidians, oocysts-forming apicomplexan protozoa, which complete their life cycle both in humans and animals, through zoonotic and anthroponotic transmission, causing cryptosporidiosis. The global burden of this disease is still underascertained, due to a conundrum transmission modality, only partially unveiled, and on a plethora of detection systems still inadequate or only partially applied for worldwide surveillance. In children, cryptosporidiosis encumber is even less recorded and often misidentified due to physiological reasons such as early-age unpaired immunological response. Furthermore, malnutrition in underdeveloped countries or clinical underestimation of protozoan etiology in developed countries contribute to the underestimation of the worldwide burden. Principal key indicators of the parasite distribution were associated to environmental (e.g., geographic and temporal clusters, etc.) and host determinants of the infection (e.g., age, immunological status, travels, community behaviours). The distribution was geographically mapped to provide an updated picture of the global parasite ecosystems. The present paper aims to provide, by a critical analysis of existing literature, a link between observational epidemiological records and new insights on public health, and diagnostic and clinical impact of cryptosporidiosis.

Gut microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients unveiled by an integrated meta-omics-based approach

There is evidence that nonalcoholic fatty liver disease (NAFLD) is affected by gut microbiota. Therefore, we investigated its modifications in pediatric NAFLD patients using targeted metagenomics and metabolomics. Stools were collected from 61 consecutive patients diagnosed with nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), or obesity and 54 healthy controls (CTRLs), matched in a case‐control fashion. Operational taxonomic units were pyrosequenced targeting 16S ribosomal RNA and volatile organic compounds determined by solid‐phase microextraction gas chromatography‐mass spectrometry. The α‐diversity was highest in CTRLs, followed by obese, NASH, and NAFL patients; and β‐diversity distinguished between patients and CTRLs but not NAFL and NASH. Compared to CTRLs, in NAFLD patients Actinobacteria were significantly increased and Bacteroidetes reduced. There were no significant differences among the NAFL, NASH, and obese groups. Overall NAFLD patients had increased levels of Bradyrhizobium, Anaerococcus, Peptoniphilus, Propionibacterium acnes, Dorea, and Ruminococcus and reduced proportions of Oscillospira and Rikenellaceae compared to CTRLs. After reducing metagenomics and metabolomics data dimensionality, multivariate analyses indicated a decrease of Oscillospira in NAFL and NASH groups and increases of Ruminococcus, Blautia, and Dorea in NASH patients compared to CTRLs. Of the 292 volatile organic compounds, 26 were up‐regulated and 2 down‐regulated in NAFLD patients. Multivariate analyses found that combination of Oscillospira, Rickenellaceae, Parabacteroides, Bacteroides fragilis, Sutterella, Lachnospiraceae, 4‐methyl‐2‐pentanone, 1‐butanol, and 2‐butanone could discriminate NAFLD patients from CTRLs. Univariate analyses found significantly lower levels of Oscillospira and higher levels of 1‐pentanol and 2‐butanone in NAFL patients compared to CTRLs. In NASH, lower levels of Oscillospira were associated with higher abundance of Dorea and Ruminococcus and higher levels of 2‐butanone and 4‐methyl‐2‐pentanone compared to CTRLs. Conclusion: An Oscillospira decrease coupled to a 2‐butanone up‐regulation and increases in Ruminococcus and Dorea were identified as gut microbiota signatures of NAFL onset and NAFL‐NASH progression, respectively. (Hepatology 2017;65:451‐464)

The human gut microbiota: a dynamic interplay with the host from birth to senescence settled during childhood

The microbiota “organ” is the central bioreactor of the gastrointestinal tract, populated by a total of 1014 bacteria and characterized by a genomic content (microbiome), which represents more than 100 times the human genome. The microbiota plays an important role in child health by acting as a barrier against pathogens and their invasion with a highly dynamic modality, exerting metabolic multistep functions and stimulating the development of the host immune system, through well-organized programming, which influences all of the growth and aging processes. The advent of “omics” technologies (genomics, proteomics, metabolomics), characterized by complex technological platforms and advanced analytical and computational procedures, has opened new avenues to the knowledge of the gut microbiota ecosystem, clarifying some aspects on the establishment of microbial communities that constitute it, their modulation and active interaction with external stimuli as well as food, within the host genetic variability. With a huge interdisciplinary effort and an interface work between basic, translational, and clinical research, microbiologists, specialists in “-omics” disciplines, and clinicians are now clarifying the role of the microbiota in the programming process of several gut-related diseases, from the physiological symbiosis to the microbial dysbiosis stage, through an integrated systems biology approach.

Gut microbiota profiling: Metabolomics based approach to unravel compounds affecting human health

The gut microbiota is composed of a huge number of different bacteria, that produce a large amount of compounds playing a key role in microbe selection and in the construction of a metabolic signaling network. The microbial activities are affected by environmental stimuli leading to the generation of a wide number of compounds, that influence the host metabolome and human health. Indeed, metabolite profiles related to the gut microbiota can offer deep insights on the impact of lifestyle and dietary factors on chronic and acute diseases. Metagenomics, metaproteomics and metabolomics are some of the meta-omics approaches to study the modulation of the gut microbiota. Metabolomic research applied to biofluids allows to: define the metabolic profile; identify and quantify classes and compounds of interest; characterize small molecules produced by intestinal microbes; and define the biochemical pathways of metabolites. Mass spectrometry and nuclear magnetic resonance spectroscopy are the principal technologies applied to metabolomics in terms of coverage, sensitivity and quantification. Moreover, the use of biostatistics and mathematical approaches coupled with metabolomics play a key role in the extraction of biologically meaningful information from wide datasets. Metabolomic studies in gut microbiota-related research have increased, focusing on the generation of novel biomarkers, which could lead to the development of mechanistic hypotheses potentially applicable to the development of nutritional and personalized therapies.

Mediterranean Diet and Health: Food Effects on Gut Microbiota and Disease Control

The Mediterranean diet (MD) is considered one of the healthiest dietary models. Many of the characteristic components of the MD have functional features with positive effects on health and wellness. The MD adherence, calculated through various computational scores, can lead to a reduction of the incidence of major diseases (e.g., cancers, metabolic and cardiovascular syndromes, neurodegenerative diseases, type 2 diabetes and allergy). Furthermore, eating habits are the main significant determinants of the microbial multiplicity of the gut, and dietary components influence both microbial populations and their metabolic activities from the early stages of life. For this purpose, we present a study proposal relying on the generation of individual gut microbiota maps from MD-aware children/adolescents. The maps, based on meta-omics approaches, may be considered as new tools, acting as a systems biology-based proof of evidence to evaluate MD effects on gut microbiota homeostasis. Data integration of food metabotypes and gut microbiota “enterotypes” may allow one to interpret MD adherence and its effects on health in a new way, employable for the design of targeted diets and nutraceutical interventions in childcare and clinical management of food-related diseases, whose onset has been significantly shifted early in life.

Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25–28S rRNA gene

Clinically relevant yeasts are conventionally identified by a combination of phenotypic tests, which occasionally provide ambiguous results for atypical isolates or uncommon species. In this study, we evaluate a direct polymerase chain reaction‐sequencing method, which exploits sequence divergence in the hypervariable D2 region of the large subunit of the 25–28S ribosomal RNA (rRNA) gene for identification of facultative pathogenic asco‐ and basidiomycota. A panel of 53 yeasts, including 40 clinical isolates and 13 reference strains representative of some clinically relevant taxa, was investigated by combining standard phenotypic tests with commercial identification systems (RapID, API 20C AUX), and results were compared with the taxonomic allocations inferred by D2 sequence analysis. Species‐level resolution was achieved for almost all (52/53) strains by combining internet‐based D2 sequence homology (BLAST and FASTA) searches in free‐access synchronised databases with phylogenetic analysis. The phylogenetic information carried by the short D2 sequence substantiates a pattern of molecular evolution, which is similar to that inferred from analysis of the larger D1/D2 region, and consistent with previously published 25–28S rRNA phylogenetic architectures of facultative pathogenic yeast, including recently identified species. Inconsistency between conventional and molecular identification results was observed for 11/53 strains, likely on account of the ambiguous interpretation of phenotypic tests.

Investigation of Toxoplasma gondii presence in farmed shellfish by nested-PCR and real-time PCR fluorescent amplicon generation assay (FLAG)

To evaluate the presence of Toxoplasma gondii in edible farmed shellfish, 1734 shellfish specimens i.e., 109 Crassostrea gigas (6 pools), 660 Mytilus galloprovincialis (22 pools), 804 Tapes decussatus (28 pools) and 161 Tapes philippinarum (6 pools), were collected from the Varano Lagoon (Apulia, Italy). Shellfish from 62 pools were subjected to two molecular techniques: a nested-PCR assay, and a fluorescent amplicon generation (FLAG) real-time PCR assay, both based on the multi-copy B1 target, were performed. One pooled sample of gills from C. gigas and one pooled sample of haemolymphs from T. decussatus were assessed as positive for T. gondii DNA by both techniques. The results demonstrated the presence of T. gondii in edible farmed C. gigas and T. decussatus and indicate that there may be a considerable health threat involved in eating contaminated raw shellfish.

Involvement of AlgQ in Transcriptional Regulation of Pyoverdine Genes in Pseudomonas aeruginosa PAO1

In response to iron limitation, Pseudomonas aeruginosa produces the fluorescent siderophore pyoverdine. Transcription of pyoverdine biosynthetic (pvd) genes is driven by the iron starvation sigma factor PvdS, which is negatively regulated by the Fur-Fe(II) holorepressor. We studied the effect of AlgQ, the Escherichia coli Rsd orthologue, on pyoverdine production by P. aeruginosa PAO1. AlgQ is a global regulatory protein which activates alginate, ppGpp, and inorganic polyphosphate synthesis through a cascade involving nucleoside diphosphate kinase (Ndk). AlgQ is also capable of interacting with region 4 of RpoD. In a reconstituted E. coli system, PvdS-dependent transcription from the pvdA promoter was doubled by the multicopy algQ gene. The P. aeruginosa DeltaalgQ mutant exhibited a moderate but reproducible reduction in pyoverdine production compared with wild-type PAO1, as a result of a decline in transcription of pvd genes. PvdS expression was not affected by the algQ mutation. Single-copy algQ fully restored pyoverdine production and expression of pvd genes in the DeltaalgQ mutant, while ndk did not. An increased intracellular concentration of RpoD mimicked the DeltaalgQ phenotype, whereas PvdS overexpression suppressed the algQ mutation. E. coli rsd could partially substitute for algQ in transcriptional modulation of pvd genes. We propose that AlgQ acts as an anti-sigma factor for RpoD, eliciting core RNA polymerase recruitment by PvdS and transcription initiation at pvd promoters. AlgQ provides a link between the pyoverdine and alginate regulatory networks. These systems have similarities in responsiveness and physiological function: both depend on alternative sigma factors, respond to nutrient starvation, and act as virulence determinants for P. aeruginosa.

Phylogenetic and Metabolic Tracking of Gut Microbiota during Perinatal Development

The colonization and development of gut microbiota immediately after birth is highly variable and depends on several factors, such as delivery mode and modality of feeding during the first months of life. A cohort of 31 mother and neonate pairs, including 25 at-term caesarean (CS) and 6 vaginally (V) delivered neonates (DNs), were included in this study and 121 meconium/faecal samples were collected at days 1 through 30 following birth. Operational taxonomic units (OTUs) were assessed in 69 stool samples by phylogenetic microarray HITChip and inter- and intra-individual distributions were established by inter-OTUs correlation matrices and OTUs co-occurrence or co-exclusion networks. 1H-NMR metabolites were determined in 70 stool samples, PCA analysis was performed on 55 CS DNs samples, and metabolome/OTUs co-correlations were assessed in 45 CS samples, providing an integrated map of the early microbiota OTUs-metabolome. A microbiota “core” of OTUs was identified that was independent of delivery mode and lactation stage, suggesting highly specialized communities that act as seminal colonizers of microbial networks. Correlations among OTUs, metabolites, and OTUs-metabolites revealed metabolic profiles associated with early microbial ecological dynamics, maturation of milk components, and host physiology.