Human microbiota-associated (HMA) models are used to allow in vivo studies of the human gut microbiome and its effects on host physiology. In particular, alterations in early life microbiota have been linked to allergy development during childhood. In this study, we investigated how pools of human microbiota collected from infants with different allergy risk, thrive in mice and their offspring, as well as how they influence the host metabolome. We used a two-generation HMA mouse model in which dams were colonized with human feces from three groups of infants (n = 19, samples collected during the first 8 weeks of life). In two of the groups, all infants had a strong hereditary risk for allergic disease (n = 12), but only 6 of them developed allergy before 2 years of age. In the third group, which was used as a control, none of the infants had allergic heredity or developed allergy (n = 7). Microbiota trajectories were followed from inoculation to mouse offspring, and metabolic profiles were monitored in several intestinal organs as well as in the serum of the murine offspring. The human microbiota adapted to the murine host but still presented distinct compositional features, reflecting the original inoculated samples. These microbial differences were mirrored in the mouse offspring metabolome, with group-associated patterns in sphingolipids, acylcarnitines and tryptophan metabolites. Furthermore, the metabolic profiles of the mouse offspring aligned with those observed in fecal water preparations from the corresponding human infant fecal samples. Our findings highlight the significant impact of early-life microbiota on the host metabolome and show that our two-generation HMA model is suitable for studying microbiota‒metabolome relationships relevant to humans. The differences in microbiota‒metabolome correlations between individuals who develop or do not develop allergic disease suggest that an allergic predisposition might be more multifaceted than previously believed.

Circulating Cathelicidin Antimicrobial Peptide LL-37 Levels in Mothers and Preterm and Term Newborn Infants.

In aquatic ecosystems, microplastics are a relatively new anthropogenic substrate that can readily be colonized by biofilm-forming organisms. To examine the effects of substrate type on microbial community assembly, we exposed ambient Baltic bacterioplankton to plastic substrates commonly found in marine environments (polyethylene, polypropylene and polystyrene) as well as native (cellulose) and inert (glass beads) particles for 2 weeks under controlled conditions. The source microbial communities and those of the biofilms were analyzed by Illumina sequencing of the 16S rRNA gene libraries. All biofilm communities displayed lower diversity and evenness compared with the source community, suggesting substrate-driven selection. Moreover, the plastics-associated communities were distinctly different from those on the non-plastic substrates. Whereas plastics hosted greater than twofold higher abundance of Burkholderiales, the non-plastic substrates had a significantly higher proportion of Actinobacteria and Cytophagia. Variation in the community structure, but not the cell abundance, across the treatments was strongly linked to the substrate hydrophobicity. Thus, microplastics host distinct bacterial communities, at least during early successional stages.

The intestinal microbiota influences immune maturation during childhood, and is implicated in early-life allergy development. However, to directly study intestinal microbes and gut immune responses in infants is difficult. To investigate how different types of early-life gut microbiota affect immune development, we collected fecal samples from children with different allergic heredity (AH) and inoculated germ-free mice. Immune responses and microbiota composition were evaluated in the offspring of these mice. Microbial composition in the small intestine, the cecum and the colon were determined by 16S rRNA sequencing. The intestinal microbiota differed markedly between the groups of mice, but only exposure to microbiota associated with AH and known future allergy in children resulted in a T helper 17 (Th17)-signature, both systemically and in the gut mucosa in the mouse offspring. These Th17 responses could be signs of a particular microbiota and a shift in immune development, ultimately resulting in an increased risk of allergy.

Background and aims Device-related infections are thought to be initiated by adhesion of the bacteria to a medical device, followed by colonisation and mature biofilm formation. Preterm infants are susceptible to device-related infections caused by Staphylococcus epidermidis. Also, preterm infants have lower levels of antimicrobial peptides, including human cathelicidin antimicrobial peptide LL37, a condition that in part may explain their increased vulnerability. Our aim was to evaluate the effect of peptide LL37 on 1) the expression of biofilm-associated genes and 2) biofilm mass, by using an in vitro model. Methods Biofilm formation of S. epidemidis was studied on intra vascular catheter pieces and in culture plates, in the absence or presence of LL37. Bacterial biofilm mass was investigated by scanning electron microscopy (SEM). Changes in biofilm-associated gene expression was determined by real-time polymerase chain reaction. Results Tissue-like concentration of the peptide down-regulated most of the investigated genes after 2 h. A diminished biofilm mass was seen on the catheter surface by SEM after 24 h incubation. Conclusions Peptide LL37, as part of innate immune defense of the newborn infant is crucial for the regulation of the commensal flora, including Staphylococcus epidermidis. A diminished activity of LL37, as found in preterm infants, may contribute to increase their risk of device-related infections.

Staphylococcus epidermidis, a human commensal, is an important opportunistic, biofilm-forming pathogen and the main cause of late onset sepsis in preterm infants, worldwide. In this study we describe the characteristics of S. epidermidis strains causing late onset (>72 h) bloodstream infection in preterm infants and skin isolates from healthy newborns. Attachment and biofilm formation capability were analyzed in microtiter plates and with transmission electron microscopy (TEM). Clonal relationship among strains was studied with pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was performed, as well as the detection of biofilm-associated genes and of the invasiveness marker IS256 with polymerase chain reaction. Blood and skin isolates had similar attachment and biofilm-forming capabilities and biofilm formation was not related to the presence of specific genes. Filament-like membrane structures were seen by TEM early in the attachment close to the device surface, both in blood and skin strains. Nine of the ten blood isolates contained the IS256 and were also resistant to methicillin and gentamicin in contrast to skin strains. S. epidermidis strains causing bloodstream infection in preterm infants exhibit higher antibiotic resistance and are provided with an invasive genetic equipment compared to skin commensal strains. Adhesion capability to a device surface seems to involve bacterial membrane filaments.

Staphylococcus epidermidis are important opportunistic biofilm forming pathogens, particularly causing infection in patients with indwelling medical devices. Preterm infants represent a high-risk group for device-related S. epidermidis infections since they require the delivery of nutrients and drugs over long periods. The present study compared genetic and phenotypic characteristics of S. epidermidis strains isolated from blood stream infections of preterm infants (n=10) versus skin isolates obtained from healthy newborns (n=16). Two reference strains were also included the study. Insertion element IS256, as a marker for invasiveness, was analysed by PCR. Antimicrobial susceptibility was testing against cefoxitin, gentamicin and vancomycin. Pulsed-Field Gel Electrophoresis was performed to study clonal relationship among strains. 90% of the blood isolates were resistant to cefoxitin and gentamicin and all these carried IS256. All skin isolates were susceptible to both cefoxitin and gentamicin and all lacked IS256. All of the 28 strains included in the study were susceptible to vancomycin. We conclude that the S. epidermidis strains isolated from blood stream infection in preterm infants are clonally not related to the normal colonizing S. epidermidis skin flora at birth, have different phenotypic features related to antimicrobial susceptibility, and have most probably originated from the hospital environment.

Aims:The aim of this work was to investigate the possible effect of human cathelicidin antimicrobial peptide LL37 on biofilm formation of Staphylococcus epidermidis, a major causative agent of indwelling device-related infections.Methods and Results:We performed initial attachment assay and biofilm formation solid surface assay in microtitre plates, as well as growth experiment in liquid medium using laboratory strain Staph. epidermidis ATCC35984. We found that already a low concentration of the peptide LL37 (1 mg l-1) significantly decreased both the attachment of bacteria to the surface and also the biofilm mass. No growth inhibition was observed even at 16 mg l-1 concentration of LL37, indicating a direct effect of the peptide on biofilm production.Conclusions:As biofilm protects bacteria during infections in humans and allows their survival in a hostile environment, inhibition of biofilm formation by LL37 may have a key role to prevent bacterial colonization on indwelling devices.Significance and Impact of the Study:Our findings suggest that this host defence factor can be a potential candidate in prevention and treatment strategies of Staph. epidermidis infections in humans.

Background: Staphylococcus epidermidis is the most commonly isolated etiological agent of nosocomial infections in preterm infants and the main cause of device-related infections. Biofilm formation is its best known virulence factor. Methods: S. epidermidis strains were isolated from normal skin and blood of preterm infants. Biofilmand initial attachment assays were determined microplates by crystal violet staining as well as on central venous catheter (CVC) visualised by transmission electron microscopy (TEM). Biofilmassociated genes, and the insertion sequence element IS256 were detected by polymerase chain reaction. The autolysin/adhesin atlE genes of isolates were sequenced. Results: TEM revealed fimbrial structures on the bacterial surface involved at the initial attachment to CVC of a skin isolate. Similarity in the attachment of the isolates was confirmed by cluster analysis of the atlE gene sequences. Biofilm-positive phenotype was a frequent finding in skin but not in blood isolates. No correlation was found between biofilm formation and presence or absence of different biofilm related genes. 9 from10 blood isolates found to be invasive based on the presence of IS256. Conclusions: Although biofilm formation is a well known virulence factor of S. epidermidis, invasive strains may use an other strategy to be in hiding from the immune system. Since IS256 may affect the expression of certain genes and also may be associated with the antibiotic resistance, these can increase the fitness of invasive strains to cause sepsis in preterms.

Coagulase-negative staphylococci and its subtype Staphylococcus epidermidis are major indigenous Gram-positive inhabitants of the human skin. Colonization occurs in direct connection with birth and terrestrial adaptation. This study focuses on factors that may influence skin colonization of the newborn infant that relates to the immune status of both the bacteria and the host. Skin is an effective barrier against bacteria, and this function is partly mediated by the presence of antimicrobial peptides including human cathelicidin peptide LL37. Gram-positive bacteria have been described to have adhesive pili on their surface that mediates specific attachment to the host. Here, we identify, by negative staining transmission electron microscopy (EM), two different types of pilus-like structures commonly expressed on S. epidermidis isolated from newborn infants. We also show that the cathelicidin antimicrobial peptide LL37, constitutively expressed in the skin barrier of the newborn, significantly inhibited growth of S. epidermidis indicating its importance for the ecological stability of the skin microbiota. Further studies are required to elucidate molecular mechanisms of host-microbe interactions, both for the maintenance of a mutually beneficial homeostatic relationship and for the protection of self when it results in overt disease.

Wohlfahrtia magnifica (Diptera: Sarcophagidae) is the major myiasis-causing fly species in the whole of Eurasia for most important domestic animals. The aim of the present work was to obtain data on the culturable bacteria isolated under aerobic conditions from this fly: bacteria were isolated from all developmental stages (larvae, pupa, and imago) of Wohlfahrtia magnifica, and the third-stage larval organs were also sampled. To determine the possible antagonistic effects between the dominant bacterial groups, an antibiosis assay was carried out. Plating and isolation of bacteria was performed by classical microbiological methods. Characterization of the isolated strains was carried out via a polyphasic approach; classical phenotypic tests, chemotaxonomical examinations, and 16S rDNA sequence analyses were also applied. In the case of maggot macerate samples, members of the family Enterobacteriaceae were characteristic. Members of a new genus (Schineria) belonging to the γ subdivision of proteobacteria were also isolated. According to our data, the shifts in the Schineria and Proteus populations within the larvae are strongly influenced by their interactions with each other and among the members of the family Enterobacteriaceae. The pupa and imago samples contained several other Gram-negative bacteria (Stenotrophomonas, Brevundimonas, etc.). Among Gram-positive bacteria, in all maggot macerate samples, members of the genus Bacillus and the Arthrobacter–Micrococcus group of actinobacteria were dominant (neither of them was a producer or sensitive to the compounds of other microorganisms), and bacteria related to the genus Corynebacterium were also found. From the larvae Aureobacterium liquefaciens and Enterococcus faecalis were isolated, and from the pupae Dietzia maris and Enterococcus faecalis. In the samples of third-stage larval organs, the dominant groups were the same as in the third-stage larval macerate sample; however, several additional genera/species were observed (Rhodococcus fascians, Streptomyces sp., Rathayibacter sp., Bacillus thuringiensis/cereus).

The species of subfamily Cyathostominae (Nematoda: Strongylidae), called as small strongyles are the most common nematodes of horses worldwide. The majority of anthelmintics are ineffective or partly effective against the mucosal larval stages of these parasites which can cause larval cyathostominosis (larval cyathostomosis) resulting in mild or severe clinical signs, mainly in chronic diarrhoea. The other problem caused by this parasitosis is related to anthelmintic resistance developed in cyathostomes against benzimidazoles and pyrantels which have been detected in many countries. For a few decades benzimidazoles and macrocyclic lactones have been used for anthelmintic treatments of horses in Hungary. However, no field or laboratory studies have evaluated the efficacy of the products against the small strongyles. For this reason the efficacy of Eqvalan paste (ivermectin), Equest 2% oral gel (moxidectin), Promectin Plus paste (abamectin and praziquantel) and PALO gel (mebendazole) was checked with an in vivo method (Fecal Egg Count Reduction Test=FECRT). Ten horses of each group infected with small strongyles were treated once with a product and ten animals of the fifth group remained untreated as controls. According to the results of FECRT, the efficacy of products containing ivermectin or moxidectin was 99% (confidence interval at the level of 95% was between 93 and 100) and 100% for abamectin. The mean fecal egg count reduction was only 18% in horses treated with PALO gel indicating that a high level of mebendazole resistance occurred in the local cyathostome populations. Based on the first detection of benzimidazole-resistant small strongyles in Hungary the authors discuss the risk factors for the development of anthelmintic resistance and those issues that should be taken into account for delaying the development of drug resistance in equine cyathostomin nematodes.

Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae) is the main agent of traumatic myiasis in many European, African and Asian countries. Although it can be reared in vivo without technical difficulty, such rearing presents ethical problems. Studies were therefore made of in vitro rearing to facilitate development of laboratory colonies that could be used in a wide range of biological, physiological and applied studies of W. magnifica, particularly in the long period of the year when natural populations of the fly are unavailable for study. Parental colonies of W. magnifica were established from larvae collected from natural infestations of sheep and cattle in central Hungary. First stage larvae were harvested from gravid females and were reared in groups of 5-20 on one of six artificial diets. The diets were based on various combinations of five to seven of eight ingredients: water, agar, blood (heparinized or dried), ground meat, egg yolk, low-fat milk powder, yeast and 10% formol. The larvae were incubated on the diets at 37 degrees C. There was no mortality of first stage larvae, which appeared to feed together in foci, in a natural manner. However, during the second stage, and especially after renewal of diet associated with disturbance of the larvae, many larvae began to disperse, crawling over the surface of the media and feeding less intensively. Mortality of larvae during all larval stadia was 64-98%, compared to 33% in batches of third stage larvae collected from natural infestations. The mean weights of puparia from artificial diets ranged from 38.7 to 59.3 mg, compared to 92.2 mg of puparia from larvae collected from natural infestations. There was a high mortality in the pupal stage, from 61 to 100%. Only a maximum of 6% of first stage larvae were successfully reared to the adult stage. Further studies are needed to identify factors present or absent in the diets that contributed to the present poor development of W. magnifica in vitro.