Bacterial Colonization of the Hospitalized Newborn: Competition Between Staphylococcus aureus and Staphylococcus epidermidis.

BACKGROUND: In adults, Staphylococcus epidermidis and Staphylococcus aureus compete for colonization of the nasal mucosa and S. epidermidis strains that produce the Esp serine protease eradicate S. aureus nasal colonization. Whether similar phenomena are seen in newborn infants is unknown. METHODS: Nasal swabs were obtained on admission and discharge from newborn infants (n = 90 and 83, respectively) in the neonatal intensive care unit at UC Davis Children's Hospital. Swabs were cultured for S. aureus and S. epidermidis. S. epidermidis isolates were tested for Esp expression, overall secreted protease activity and biofilm inhibition. RESULTS: No infant had S. aureus on admission. S. epidermidis colonization was rare on admission in inborn infants (2.5%), but common in infants transferred from referring hospitals (50%). At discharge, most infants (96%) were colonized by staphylococci. S. aureus colonization was less common in infants with S. epidermidis colonization (9%) and more common in infants without S. epidermidis (77%) (relative risk of S. aureus colonization in infants colonized with S. epidermidis 0.18, 95% confidence interval: 0.089-0.34, P < 0.0001). Compared with S. epidermidis strains from infants without S. aureus, S. epidermidis from infants co-colonized with S. aureus had lower total proteolytic enzyme activity and decreased biofilm inhibition capacity, but did not have lower frequency of Esp positivity. CONCLUSIONS: In hospitalized neonates, S. epidermidis colonization has a protective effect against S. aureus colonization. Secretion of proteases by S. epidermidis is a possible mechanism of inhibition of S. aureus colonization; however, in this cohort of neonates, the source of major protease activity is likely other than Esp.

Human ß-Defensin 2 in Primary Sclerosing Cholangitis.

OBJECTIVES: Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts frequently associated with inflammatory bowel disease (IBD), suggesting an important role for the gut-liver axis. Defensins are small (3.5-4.5 kDa) anti-microbial peptides that contribute to innate immunity at mucosal surfaces and have been implicated in IBD. The aim of this study was to investigate copy number variation of the gene (DEFB4) encoding human ß-defensin 2 (HBD2) and protein expression of HBD2 in PSC. METHODS: US and Italian PSC cases and unaffected controls (US PSC patients n=89, US controls n=87; Italian PSC patients n=46, Italian controls n=84) were used to estimate HBD2 gene copy number by both quantitative real-time PCR and paralog ratio test. Serum levels of HBD2 were measured by enzyme-linked immunosorbent assay and liver expression was analyzed by immunohistochemistry. RESULTS: Mean serum levels of HBD2 were significantly greater in PSC (1,086±1,721 ng/µl) compared with primary biliary cholangitis (544±754 ng/µl), ulcerative colitis (417±506 ng/µl), and healthy controls (514±731 ng/µl) (P=0.02). However, no significant differences between the frequencies of high DEFB4 gene copy number, defined by >4 copies, and PSC were found in the US, Italian, or combined cohorts. Importantly, a high number of biliary ducts were found immunopositive in PSC samples compared with controls. CONCLUSIONS: Our data show that HBD2 serum levels and tissue expression are increased in PSC subjects, suggesting that this arm of innate immunity may be important in the etiopathogenesis of PSC.

Bifidobacterium longum subsp. infantis in experimental necrotizing enterocolitis: alterations in inflammation, innate immune response, and the microbiota.

BACKGROUND: Probiotics decrease the risk of necrotizing enterocolitis (NEC). We sought to determine the impact of Bifidobacterium longum subsp. infantis (B. infantis) in the established rat model of NEC. METHODS: Rat pups delivered 1 d prior to term gestation were assigned to one of three groups: dam fed (DF), formula fed (FF), or fed with formula supplemented with 5 × 10(6) CFU B. infantis per day (FF+Binf). Experimental pups were exposed to hypoxia and cold stress. Ileal tissue was examined for pathology and expression of inflammatory mediators, antimicrobial peptides, and goblet-cell products. Ceca were assessed for bacterial composition by analysis of the 16S rRNA sequence. RESULTS: Administration of B. infantis significantly reduced the incidence of NEC, decreased expression of Il6, Cxcl1, Tnfa, Il23, and iNOS, and decreased expression of the antimicrobial peptides Reg3b and Reg3g. There was significant microbial heterogeneity both within groups and between experiments. The cecal microbiota was not significantly different between the FF and FF+Binf groups. Bifidobacteria were not detected in the cecum in significant numbers. CONCLUSION: In the rat model, the inflammation associated with NEC was attenuated by administration of probiotic B. infantis. Dysbiosis was highly variable, precluding determination of the precise role of the microbiota in experimental NEC.

Copy number variation of the beta defensin gene cluster on chromosome 8p influences the bacterial microbiota within the nasopharynx of otitis-prone children.

As there is increasing evidence that aberrant defensin expression is related to susceptibility for infectious disease and inflammatory disorders, we sought to determine if copy number of the beta-defensin gene cluster located on chromosome 8p23.1 (DEFB107, 106, 105, 104, 103, DEFB4 and SPAG11), that shows copy number variation as a block, was associated with susceptibility to otitis media (OM). The gene DEFB103 within this complex encodes human beta defensin-3 (hBD-3), an antimicrobial peptide (AP) expressed by epithelial cells that line the mammalian airway, important for defense of mucosal surfaces and previously shown to have bactericidal activity in vitro against multiple human pathogens, including the three that predominate in OM. To this end, we conducted a retrospective case-control study of 113 OM prone children and 267 controls aged five to sixty months. We identified the copy number of the above defined beta-defensin gene cluster (DEFB-CN) in each study subject by paralogue ratio assays. The mean DEFB-CN was indistinguishable between subjects classified as OM prone based on a recent history of multiple episodes of OM and control subjects who had no history of OM (4.4 ± 0.96 versus 4.4 ± 1.08, respectively: Odds Ratio [OR]: 1.16 (95% CI: 0.61, 2.20). Despite a lack of direct association, we observed a statistically significant correlation between DEFB-CN and nasopharyngeal bacterial colonization patterns. Collectively, our findings suggested that susceptibility to OM might be mediated by genetic variation among individuals, wherein a DEFB-CN less than 4 exerts a marked influence on the microbiota of the nasopharynx, specifically with regard to colonization by the three predominant bacterial pathogens of OM.

Helicobacter hepaticus increases intestinal injury in a rat model of necrotizing enterocolitis.

Enterohepatic helicobacter species (EHS) infect the intestinal tract and biliary tree, triggering intestinal and hepatic disorders. Helicobacter hepaticus, the prototypic murine EHS, is also associated with inflammation. Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. The cause of NEC is not fully understood, but anomalies of bacterial colonization (dysbiosis) are thought to play an important role in disease onset. To evaluate the effect of H. hepaticus infection on the development of NEC, premature formula-fed rats were kept either in H. hepaticus-free conditions or colonized with H. hepaticus; both groups were exposed to asphyxia and cold stress. The incidence of NEC, expression of Toll-like receptors (TLRs), production of cytokines and mucins, and presence of autophagy regulators were evaluated at the site of injury. H. hepaticus infection increased the incidence of NEC from 39 to 71% and significantly increased levels of TLR4 receptor, expression of proinflammatory cytokines CXCL1, IL-1ß, IL-12, and IL-23, and altered activation of autophagy. H. hepaticus induces inflammation and increases the incidence and severity of experimental NEC; this is consistent with observations in neonates of blooms of proinflammatory microbes just before the onset of NEC. Future studies using rodent NEC models should include testing for H. hepaticus infection. Further studies in neonates of early identification and/or diminution of proinflammatory microbes may be beneficial in decreasing the incidence of NEC.

Routine habitat change: a source of unrecognized transient alteration of intestinal microbiota in laboratory mice.

The mammalian intestine harbors a vast, complex and dynamic microbial population, which has profound effects on host nutrition, intestinal function and immune response, as well as influence on physiology outside of the alimentary tract. Imbalance in the composition of the dense colonizing bacterial population can increase susceptibility to various acute and chronic diseases. Valuable insights on the association of the microbiota with disease critically depend on investigation of mouse models. Like in humans, the microbial community in the mouse intestine is relatively stable and resilient, yet can be influenced by environmental factors. An often-overlooked variable in research is basic animal husbandry, which can potentially alter mouse physiology and experimental outcomes. This study examined the effects of common husbandry practices, including food and bedding alterations, as well as facility and cage changes, on the gut microbiota over a short time course of five days using three culture-independent techniques, quantitative PCR, terminal restriction fragment length polymorphism (TRFLP) and next generation sequencing (NGS). This study detected a substantial transient alteration in microbiota after the common practice of a short cross-campus facility transfer, but found no comparable alterations in microbiota within 5 days of switches in common laboratory food or bedding, or following an isolated cage change in mice acclimated to their housing facility. Our results highlight the importance of an acclimation period following even simple transfer of mice between campus facilities, and highlights that occult changes in microbiota should be considered when imposing husbandry variables on laboratory animals.

Bifidobacterium bifidum in a rat model of necrotizing enterocolitis: antimicrobial peptide and protein responses.

INTRODUCTION: Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. Probiotics decrease the risk of NEC in clinical and experimental studies. Antimicrobial peptides protect the gut against noxious microbes and shape the commensal microbiota, but their role in NEC remains unclear. METHODS: To investigate the expression of antimicrobial peptides in experimental NEC and the impact of probiotics on their expression, premature rats were divided into three groups: dam fed (DF), hand fed with formula (FF), or hand fed with formula containing Bifidobacterium bifidum (FF + BIF). All groups were exposed to asphyxia and cold stress. RESULTS: Like in human ontogeny, the rat pup has low expression of Paneth cell antimicrobials, which increases rapidly during normal development. The expression of lysozyme, secretory phospholipase A(2) (sPLA(2)), pancreatic-associated proteins 1 and 3 mRNA was elevated in the FF group with a high incidence of NEC, as compared with the DF and FF + BIF groups where the disease was attenuated. DISCUSSION: We conclude that induction of antimicrobial peptides occurs in experimental NEC similar to that reported in human disease and is attenuated when disease is averted by probiotic B. bifidum. The induction of antimicrobial peptides is likely an adaptive mucosal response that is often not sufficient to prevent disease in the premature gut.

Activity, expression and genetic variation of canine ß-defensin 103: a multifunctional antimicrobial peptide in the skin of domestic dogs.

The skin functions as more than a physical barrier to infection. Epithelial cells of the skin can synthesize antimicrobial peptides, including defensins, which exhibit direct antimicrobial activity. Here we characterize the expression pattern, genetic variation and activity of the major ß-defensin expressed in canine skin, canine ß-defensin 103 (CBD103). The gene encoding CBD103 exhibits two forms of polymorphism: a common 3-basepair deletion allele and a gene copy-number variation. Golden retrievers and Labrador retrievers were the only breeds that encoded the variant allele of CBD103, termed CBD103ΔG23. Both these breeds also exhibited a CBD103 gene copy-number polymorphism that ranged from 2 to 4 gene-copies per diploid genome. Recombinant CBD103 and CBD103ΔG23, as well as the human ortholog human ß-defensin 3 (hBD3) and hBD3ΔG23, showed potent and comparable antimicrobial killing against both methicillin-susceptible and methicillin-resistant Staphylococcus pseudintermedius. Skin biopsy specimens from dogs with atopic dermatitis revealed CBD103 expression levels similar to those in healthy controls and comparable at lesional and nonlesional sites. This expression pattern in dogs differs from the previously reported reduced expression of the human ortholog in atopic dermatitis. Overall, the similarities of CBD103 and its human ortholog reported here support the notion that the domestic dog may serve as a valuable model for studying ß-defensin biology in the skin.

Nucleotide-binding oligomerization domain-containing protein 2 regulates suppressor of cytokine signaling 3 expression in Burkholderia pseudomallei-infected mouse macrophage cell line RAW 264.7.

Burkholderia pseudomallei, a causative agent of melioidosis, is a facultative intracellular Gram-negative bacterium that can survive and multiply inside the macrophages. Toll-like receptors are one class of pattern recognition receptors (PRRs) that have been documented to play significant role in B. pseudomallei infection. In the present study, we investigated a potential role of nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD1 and NOD2), cytoplasmic pattern recognition receptors, in B. pseudomallei-infected mouse macrophage cell line RAW 264.7. Both live and heat-killed B. pseudomallei were able to up-regulate NOD1 and NOD2 expression in a time-dependent manner. Marked reduction of a negative regulator, suppressor of cytokine signaling 3 (SOCS3), expression was observed only in B. pseudomallei-infected NOD2-depleted macrophages and not in NOD1-depleted macrophages. The decrease in SOCS3 expression also led to an increase in IFN-γ responsiveness as judged by an enhanced STAT-1 phosphorylation on tyrosine 701 in the B. pseudomallei-infected macrophages. Together, these results suggested that, in addition to using other PRRs to evade macrophage defense, B. pseudomallei may also use NOD2 to regulate a negative regulator like SOCS3.